Human monoclonal antibody against coreceptors for human immunodeficiency virus

ABSTRACT

Compositions are provided that comprise antibody against coreceptors for human immunodeficiency virus such as CCR5 and CXCR4. In particular, monoclonal human antibodies against human CCR5 are provided that bind to CCR5 with high affinity and are capable of inhibiting HIV infection at low concentrations. The antibodies can be used as prophylactics or therapeutics to prevent and treat HIV infection, for screening drugs, and for diagnosing diseases or conditions associated with interactions with HIV coreceptors.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods for generating monoclonal antibodyagainst cell membrane proteins, and, more particularly, to methods forgenerating human monoclonal antibodies against cell surface coreceptorsfor human immunodeficiency virus (HIV) and using these antibodies fordiagnostic or therapeutic purposes.

2. Description of Related Art

HIV infection has been implicated as the primary cause of the slowlydegenerate disease of the immune system termed acquired immunedeficiency syndrome (AIDS). Barre-Sinoussi et al. (1983) Science220:868-870; and Gallo et al. (1984) Science 224:500-503. Infection ofthe CD4+ subclass of T-lymphocytes with the HIV-1 virus leads todepletion of this essential lymphocyte subclass which inevitably leadsto opportunistic infections, neurological disease, neoplastic growth andeventually death. HIV-1 infection and HIV-1 associated diseasesrepresent a major health problem and considerable attention is currentlybeing directed towards the successful design of effective therapeutics.

HIV-1 is a member of the lentivirus family of retroviruses. Teich et al.(1984) In RNA Tumor Viruses ed. R. Weiss, N. Teich, H. Varmus, J. CoffinCSH Press, pp. 949-56. The life cycle of HIV-1 is characterized by aperiod of proviral latency followed by active replication of the virus.The primary cellular target for the infectious HIV-1 virus is the CD4subset of human T-lymphocytes. Targeting of the virus to the CD4 subsetof cells is due to the fact that the CD4 cell surface protein acts asthe cellular receptor for the HIV-1 virus. Dalgleish et al. (1984)Nature 312:763-67; Klatzmann (1984) Nature 312:767-68; and Maddon et al.(1986) Cell 47:333-48.

After binding to the cell surface, the HIV-1 virion becomesinternalized, and once inside the cell, the viral life cycle begins byconversion of the RNA genome into linear DNA molecules. This process isdependent on the action of the virally encoded reverse transcriptase.Following replication of the viral genome, the linear DNA moleculeintegrates into the host genome through the action of the viralintegrase protein, thus establishing the proviral form of HIV-1.

It was later discovered that other than CD4, HIV-1 utilizes several cellmembrane proteins as its coreceptor to falitate viral entry into thehost cell. Alkhatib et al. (1996) Science 272: 1955-1958; and Deng etal. (1996) Nature 388:296-300. Examples of chemokine receptors includeCXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, andCX₃CR1. Examples of chemokine receptor-like orphan proteins includeSTRL33/BONZO and GPR15/BOB.

CXCR4 (also known as “fusin”) is a receptor for chemokines such asSDF-1α and SDF-1β. CCR5 is a receptor for several CC chemokines such asMIP-1α (also named GOS19, LD78, pAT464 gene product, TY5 (murine) andSIS (murine)), MIP-1β (also named Act-2, G-26, pAT744 gene product,H-400 (murine) and hSISγ (murine)) and RANTES (regulated on activation,normal T cell expressed and secreted, or CCL5). Cocchi et al. (1995)Science 270:1811-1815. Binding of chemokines to CCR5 can induceactivation of JAK/STAT pathway. Mellado et al. (2001) Annu. Rev.Immunol. 19:397-421. The roles of these CC chemokine molecules inregulating T cell fate include possible indirect effects onantigen-presenting cells and direct effects on differentiating T cells.Luther & Cyster (2001) Nat. Immunol. 2:102-107.

Specific chemokine receptors such as CXCR4 and CCR5 receptors playimportant roles in mediating HIV entry and tropism for different targetcells. See reviews by Berger (1997) AIDS 11, Suppl. a: S3-S16; andDimitrov (1997) Cell 91: 721-730; and Burger et al. (1999) Annu. Rev.Immunol. 17:657-700. Macrophages-tropic (M-tropic) strains of HIV viruscan replicate in primary CD4⁺ T cells and macrophages and use theβ-chemokine receptor CCR5 and less often, CCR3 receptor. T cellline-tropic (T-tropic) HIV strains can also replicate in primary CD4⁺ Tcells but can in addition infect established CD4⁺ T cell lines in vitrovia the α-chemokine receptor CXCR4. Many of the T-tropic strains can useCCR5 in addition to CXCR4. Chemokine receptor-like HIV coreceptor STRL33is expressed in activated peripheral blood lymphocytes and T-cell linesand can function as an entry cofactor for Env proteins from M-tropic,T-tropic and dual tropic strains of HIV-1 and SIV. Other HIV coreceptorshave also been identified by numerous in vitro assays, includingchemokine receptors CCR2b, CCR3, CCR8 and CX3CR1 as well as severalchemokine receptor-like orphan receptor proteins such as GPR15/BOB andSTRL33/BONZO. Each or a set of these HIV coreceptors can mediate entryof different strains of HIV virus into the host cell.

The CC chemokine receptor CCR5 is a principal HIV-1 coreceptor thatplays a dominant role in disease transmission and in the early course ofinfection. Berger et al. (1999) Annu. Rev. Immunol. 17:657-700.Molecular epidemiology studies clearly demonstrated that CCR5 playscritical roles in HIV-1 transmission and pathogenesis. Individualslacking two copies of functional CCR5 alleles (Δ32 allele) are stronglyprotected against HIV-1 infection. Dean et al. (1996) Science273:1856-1862. Individuals with one Δ32 and one normal CCR5 gene onaverage express lower levels of CCR5 on their T cells. Wu et al. (1997)J. Exp Med. 185:1681-1691. Heterozygosity for the Δ32 allele does notprotect against HIV-1 infection but does confer an improved prognosis inthe form of significantly increased AIDS-free and overall survivalperiods. Husman et al. (1997) Ann. Intern. Med. 127:882-890. Moreover,CCR5 heterozygotes are overrepresented among long-term nonprogressors,i.e., those individuals who do not progress to AIDS after 10 or moreyears of infection. Dean et al. (1996) Science 273:1856-1862. Because itis an essential coreceptor for clinically relevant strains of HIV-1 andyet is apparently dispensable for human health, CCR5 provides anattractive target for new antiretroviral therapies. Liu et al. (1996)Cell 86:367-377; and Michael & Moore (1999) Nat. Med. 5:740-742.

Several approaches have been employed to target HIV coreceptors,involving proteins, peptides and small molecules. It has been found thatsome CCR5-targeting chemokines and chemokine analogs are capable ofinhibiting HIV-1 replication in vitro. Berger et al. (1999) Annu. Rev.Immunol. 17:657-700. Of the CC chemokines that bind CCR5, RANTESpossesses significantly greater breadth of antiviral activity thanMIP-1α and MIP-1β, although all CC chemokines show interisolatevariation in potency. Trkola et al. (1998) J. Viol. 72:396-404. Theantiviral activity of the CC chemokines better correlates with theirability to downregulate rather than to bind CCR5 on CD4 T cells, andsustained down-regulation of CCR5 has been suggested to be a principalmechanism of action for the chemokine analog aminooxypentane(AOP)-RANTES. Mack et al. (1998) J. Exp. Med. 187:1215-1224. A smallnon-peptide molecule designated TAK-779 was found to be an antagonistagainst CCR5 presumably through binding to a hydrophobic pocket definedby the transmembrane helices 1, 2, 3 and 7. Baba et al. (1999) Proc.Natl. Acad. Sci. USA 96:5698-5703; Shiraishi et al. (2000) J. Med. Chem.43:2049-2063; and Dragic et al. (2000) Proc. Natl. Acad. Sci. USA97:5639-5644.

Phage display has been utilized to select for single chain antibodyagainst CCR5 from a human antibody library by using CCR5-expressing CD4⁺lymphocytes as the target in the presence and absence of MIP-1α. Osbournet al. (1998) Nature Biotech. 16:778-781. The selected phages wereanalyzed by phage ELISA for their ability to recognize CD4⁺ lymphocytes,CCR5-transfected CHO cell line, non-transfected CHO cell line, and aBSA-conjugated peptide corresponding to the N-terminal 20 amino acidpeptide of CCR5. Osbourn et al. found that none of the antibodiesselected in the presence of MIP-1α blocked MIP-1α binding to CD4⁺lymphocytes. Among the antibodies selected in the absence of MIP-1α,around 20% inhibited MIP-1α binding to CD4⁺ lymphocytes, as well asMIP-1α-mediated calcium signaling.

Mouse monoclonal antibodies have also been generated to target CCR5 byusing the whole protein of CCR5 as the antigen. For example, Wu et al.immunized mice with the murine pre-B cell lymphoma cell line L1.2expressing high levels of transfected CCR5, which generated a IgG1monoclonal antibody, designated as mAb 2D7. Wu et al. (1997) J. Exp.Med. 186:1373-1381. The binding site of this monoclonal on CCR5 wasmapped to the second extracellular loop of CCR5. MAb 2D7 was shown to beable block the binding and chemotaxis of the three natural chemokineligands of CCR5, RANTES, macrophage inflammatory protein MIP-1α, andMIP-1β, to CCR5 transfectants. MAb 2D7 failed to stimulate an increasein intracellular calcium concentration in the CCR5 transfectants, butblocked calcium response elicited by RANTES, MIP-1α and MIP-1βchemotactic responses of activated T cells, but not of monocyte. Incontrast, a group of mAbs that were also generated in the same processand failed to clock chemokine binding were all mapped to the N-terminalregion of CCR5.

Using a similar strategy to Wu et al. (1997), Olson et al. isolated 6anti-CCR5 murine monoclonal antibodies (MAbs) by intraperitoneallyimmunizing female BALB/c mice with murine L1.2 cells expressing CCR5.Olson et al. (1999) J. Virol. 73:4145-4155. Epitope mapping of theseMAbs reveals that the epitopes of these antibodies reside in theN-terminus and/or second extracellular loop regions of CCR5. Thisstructural information was correlated with the antibodies' abilities toinhibit (1) HIV-1 entry; (2) HIV-1 envelope glycoprotein-mediatedmembrane fusion; (3) gp120 binding to CCR5; and (4) CC-chemokineacitvity. Surprisingly, each of the antibodies displayed distinctlydifferent activities in different stages of HIV-1 entry. In particular,one of these MAbs, PRO140, was shown to exert inhibitory effects onHIV-1 infection on primary peripheral blood mononuclear cells (PBMC).Trkola et al. (2001) J. Virol, 75:579-588.

SUMMARY OF THE INVENTION

The present invention provides innovative methods for efficient, highthroughput screening of antibody library against a wide variety ofproteins targets, especially against membrane proteins. In particular,methods are provided for screening fully human antibody library againstmembrane proteins such as HIV coreceptors in yeast. More particularly,single chain antibodies against fragments of CCR5 have been selected anddemonstrated to inhibit HIV infection at sub-nanomolar concentrations.

In one aspect, a method is provided for screening a library of singlechain antibodies (scFv) against a target peptide in yeast. In oneembodiment, the method comprising:

expressing a library of scFv fusion proteins in yeast cells, each scFvfusion protein comprising either an activation domain or a DNA bindingdomain of a transcription activator and a scFv, the scFv comprising aV_(H) of antibody whose sequence varies within the library, a V_(L) ofantibody whose sequence varies within the library independently of theV_(H), and a linker peptide which links the V_(H) and V_(L);

expressing a target fusion protein in the yeast cells expressing thescFv fusion proteins, the target fusion protein comprising either theDNA binding domain or the activation domain of the transcriptionactivator which is not comprised in the scFv fusion proteins, and atarget peptide; and

selecting those yeast cells in which a reporter gene is expressed, theexpression of the reporter gene being activated by a reconstitutedtranscriptional activator formed by binding of the scFv fusion proteinto the target fusion protein.

According to the embodiment, the diversity of the library scFv fusionproteins is preferably higher than 1×10⁴, more preferably higher than1×10⁶, and most preferably higher than 1×10⁷.

Also according to the embodiment, the length of the target peptide ispreferably 5-100 aa, more preferably 10-80 aa, and most preferably 20-60aa.

Also according to the embodiment, the target peptide may be a fragmentof a protein that includes an antigenic deteminant or epitope,preferably a fragment of a membrane protein, more preferably anextracellular domain of a membrane protein, and most preferably anextracellular loop of a transmembrane protein.

Examples of membrane proteins from which the target peptide may bederived include, but are not limited to, receptors for growth factors(e.g., vascular endothelial growth factor (VEGF), transforming growthfactor (TGF), fibroblast growth factor (FF), platelet derived growthfactor (PDGF), insulin-like growth factor), insulin receptor, MHCproteins (e.g. class I MHC and class II MHC protein), CD3 receptor, Tcell receptors, cytokine receptors such as interleukin-2 (IL-2)receptor, tyrosine-kinase-associated receptors such as Src, Yes, Fgr,Lck, Flt, Lyn, Hck, and Blk, and G-protein coupled receptors such asreceptors for the hormone relaxin (LGR7 and LGR8) and coreceptors forHIV (e.g., CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2,CXCR3, CX₃CR1, STRL33/BONZO and GPR15/BOB).

In a particular variation of the embodiment, the target peptidecomprises an extracellular domain of human CCR5 selected from the groupconsisting of N-terminal domain, loop 2, loop 4, and loop 6 of humanCCR5. Preferably, the extracellular domain of human CCR5 comprises asequence selected from the group consisting of SEQ ID Nos: 2, 3, 8, and9.

Also according to the embodiment, the activation domain or the DNAbinding domain of the transcription activator may optionally be fused toC-terminus of the scFv, or to the N-terminus of the scFv.

Also according to the embodiment, the activation domain or the DNAbinding domain of the transcription activator may optionally be fused toC-terminus of the target peptide, or to the N-terminus of the targetpeptide.

According to the embodiment, the step of expressing the library of scFvfusion proteins in yeast cells may include transforming a library ofscFv expression vectors into the yeast cells which contain the reportergene.

Optionally, the step of expressing the target fusion proteins includestransforming a target expression vector into the yeast cellssimultaneously or sequentially with the library of scFv expressionvectors.

Also according to the embodiment, the steps of expressing the library ofscFv fusion proteins and expressing the target fusion protein mayoptionally include causing mating between first and second populationsof haploid yeast cells of opposite mating types.

The first population of haploid yeast cells comprises a library of scFvexpression vectors for the library of scFv fusion proteins. The secondpopulation of haploid yeast cells comprises a target expression vector.Either the first or second population of haploid yeast cells comprisesthe reporter gene.

The haploid yeast cells of opposite mating types may preferably be α anda type strains of yeast. The mating between the first and secondpopulations of haploid yeast cells of α and a type strains may beconducted in a rich nutritional culture medium.

It should be noted that the above-described target peptide fragmentderived from a membrane protein may be screened against an antibodylibrary in other organisms or in vitro. For example, the target peptidemay be expressed as a fusion protein with another protein and screenedagainst an antibody library co-expressed in mammalian cells. The targetpeptide may also be immobilized to a substrate as a single peptide or afusion protein and selected against a library of antibodies displayed onthe surface of bacteriophage or displayed on ribosomes. In addition, thetarget peptide may be introduced to xenomice which contain a library ofhuman antibody and selected for monoclonal human antibodies withspecific binding affinity to target peptide and/or the target membraneprotein.

In another aspect of the present invention, compositions that compriseat least one of the heavy chain and light chain variable region of anantibody are provided which recognize epitopes on the extracellulardomains of human CCR5.

In one embodiment, the composition comprises an antibody that binds toloop 6 of human CCR5. In a variation, the antibody is capable ofinhibiting HIV-1 infection of human cells.

It is noted the antibody may be a polyclonal or a monoclonal antibody,including but not limited to fully assembled antibody, single chainantibody, Fab fragment, and chimeric antibody.

Optionally, CDR2 of the heavy chain variable region of the antibodycomprises amino acid sequence GSTX₁YNPSL [SEQ ID NO: 32], wherein X₁ isasparagine (N) or threonine (T).

Optionally, CDR2 of the light chain variable region of the antibodycomprises amino acid sequence DAX₂X₃L [SEQ ID NO: 33], wherein X₂ isthreonine (T) or serine (S), and X₃ is threonine (T) or aspartic acid(D).

Optionally, CDR2 of the heavy chain variable region of the antibodycomprises amino acid sequence GSTX₁YNPSL [SEQ ID NO: 32]; and CDR2 ofthe light chain variable region of the antibody comprises amino acidsequence DAX₂X₃L [SEQ ID NO: 33], wherein X₁ is asparagine (N) orthreonine (T), X₂ is threonine (T) or serine (S), and X₃ is threonine(T) or aspartic acid (D).

Optionally, CDR3 of the heavy chain variable region of the monoclonalantibody comprises 5, 6, 7, 8, 9 or more consecutive amino acids of asequence elected from the group consisting of

RLKGAWLLSEPPYFSSDGMDV [SEQ ID NO: 43],

RTVAGTSDY [SEQ ID NO: 44], and

HEQYYYDTSGQPYYFDF [SEQ ID NO: 45].

Optionally, CDR3 of the light chain variable region of the monoclonalantibody comprises 5, 6, 7, 8, 9 or more consecutive amino acids of asequence elected from the group consisting of

AAWDESLNGVV [SEQ ID NO: 46],

LQHDNFPLT [SEQ ID NO: 47], and

QQSDYLPLT [SEQ ID NO: 48].

Optionally, CDR3 of the heavy chain variable region of the monoclonalantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID Nos: 43-45; and CDR3 of the light chain variableregion of the monoclonal antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID Nos: 46-48.

It is noted that the above-described different CDR regions may all beincluded in the antibody independent of each other, or in combinationwith one or more of each other.

Optionally, CDR3 of the heavy chain variable region of the antibodycomprises an amino acid sequence selected from the group consisting ofSEQ ID Nos: 43-45; and CDR3 of the light chain variable region of theantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID Nos: 46-48.

Optionally, the heavy chain variable region of the antibody comprises anamino acid sequence selected from SEQ ID Nos: 36, 38, and 40.

Optionally, the light chain variable region of the antibody comprises anamino acid sequence selected from SEQ ID Nos: 37, 39, and 41.

The antibody of the present invention may be produced by expression inbacteria, yeast, plant, and animal cells in any form including but notlimited to single chain, Fab, full length IgA, secretion form sIgA, orIgG.

The antibody of present invention may be used for the prevention ortreatment of HIV infection. For example, the antibodies against humanCCR5 may be administered to an individual with high risk of HIVinfection or already infected with HIV to block the entry of HIV-1 intothe cells.

The antibody of present invention may also be conjugated with a moleculesuch as an antiviral drug and a radio-isotope to specifically targetcells expressing human CCR5.

The antibody of present invention may also be used in combination withother therapeutic agents such as proteins, antibodies, andantiretroviral drugs such as nucleoside or non-nucleoside HIV reversetranscriptase inhibitors, HIV protease inhibitors, and HIV integraseinhibitors.

Examples of the nucleoside HIV reverse transcriptase inhibitor include,but are not limited to zidovudine (AZT), didanosine (ddI), zalcitabine(ddC), lamivudine (3TC), stavudine (d4T), abacavir (1592U89), andadefovir dipivoxil (bis(POM)-PMEA). Examples of the non-nucleoside HIVreverse transcriptase inhibitor include, but are not limited tonevirapine (BI-RG-587), delavirdine (BHAP, U-90152) and efavirenz (DMP266).

Examples of the HIV protease inhibitors include, but are not limited toindinavir (MK-639), ritonavir (ABT-538), saqinavir (Ro-31-8959),nelfinavir (AG-1343), and amprenavir (141W94).

The antibody of the present invention may be administered to a mammal,preferably a human, via a variety of routes, including but not limitedto, orally, parenterally, intraperitoneally, intravenously,intraarterially, topically, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery (for exampleby catheter or stent), subcutaneously, intraadiposally,intraarticularly, or intrathecally. The antibody may also be deliveredto the host locally (e.g., via stents or catheters) and/or in atimed-release manner.

The antibody of the present invention may also be used for diagnosis ofdiseases associated with CCR5 interactions such as HIV. Moreover, theantibody may be used in assays for screening therapeutic agents againstthese diseases.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows the amino acid sequence of human CCR5.

FIG. 1B shows the amino acid sequences of peptide fragments derived fromhuman CCR5 that are used as target peptides for eliciting antibodyaccording to the present invention.

FIG. 1C shows and a model of the secondary structure of human CCR5.

FIG. 2A illustrates an embodiment of the method of present invention forscreening of scFv against a target peptide derived from a membraneprotein via transformation of yeast cells.

FIG. 2B illustrates another embodiment of the method of presentinvention for screening of scFv against a target peptide derived from amembrane protein via mating of two yeast strains.

FIG. 3 illustrates a method of constructing a human scFv antibodylibrary via homologous recombination in yeast.

FIG. 4 illustrates a method of affinity maturation of an antibody lead.

FIG. 5 shows DNA and amino acid sequences of four distinct scFvantibodies against human CCR5 fragments.

FIG. 6 shows DNA and amino acid sequences of variants of the four scFvantibodies against human CCR5 fragments.

FIG. 7 shows a homology alignment of amino acid sequences of three scFvantibodies against human CCR5 Loop6.

FIG. 8 shows amino acid sequences of V_(H) and V_(L) of the four scFvantibodies against human CCR5 fragments.

FIGS. 9A-C show HIV-1 reverse transcriptase (RT) activity in a cultureof human monocytes infected by HIV-1 in the present or absent ofantibody on day 4, 8, and 12 post infection, respectively.

FIGS. 10A-C show viability of a culture of human monocytes infected byHIV-1 in the present or absent of antibody on day 4, 8, and 12 postinfection, respectively.

FIGS. 11A-C show HIV-1 reverse transcriptase (RT) activity in a cultureof human monocytes infected by HIV-1 in the present or absent ofantibody at lower concentrations than those in FIGS. 9A-C on day 4, 8,and 12 post infection, respectively.

FIG. 12 shows a Western blot of CCR5 expressed by human macrophageprobed by scFv against human CCR5 Loop 6.

FIG. 13 is a graph showing that two scFv against human CCR5 Loop 6 areboth capable of blocking the binding of MIP-1α to CCR5 on humanmonocyte-derived macrophages.

FIG. 14 is a graph showing that non-labeled CCR5 ligands, MIP-1α andRANTES, can compete with radio-labeled MIP-1α in binding with CCR5 onhuman

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides innovative methods for efficient, highthroughput screening of antibody library against a wide variety oftarget proteins, especially membrane proteins, in yeast. In particular,the methods can be used to systematically and efficiently screen humanantibody against epitopes on a target protein and select for antibodieswith high affinity and efficacy in regulating the biological functionsof the target protein. More particularly, fully human antibodies can beselected by using these methods to target therapeutically significantmembrane proteins, such as cell surface co-receptors for HIV envelopeprotein (e.g., CXCR4 and CCR5).

Membrane proteins are generally considered to be evasive targets forscreening agents for therapeutic intervention and rational drug designbecause of difficulties associated with isolation and purification, aswell as the structural uncertainty of the isolated protein adopted invitro. As described in the section of “Background of the Invention”,skilled artisans resorted to using cells expressing the whole protein ofthe membrane protein such as CCR5 as an immunogen to elicit monoclonalantibody against it.

Surprisingly, the inventors discovered that peptide fragments of amembrane protein, as opposed to the whole protein, can be excellenttargets against which high affinity antibody can be selected in a yeasttwo-hybrid system. The peptide fragment is expressed as a target fusionprotein with the DNA-binding domain (BD) (or the activation domain (AD))of a transcription activator in yeast cells. A library of fully humansingle-chain antibody is expressed as tester fusion proteins with the AD(or the BD) of the transcription activator in the same yeast cells.Binding of the antibody to the peptide target triggers expression of areporter gene in the yeast cell, which facilitates identification andisolation of the clones containing the monoclonal human antibody. Theability of the selected monoclonal antibodies in blocking HIV entry andinhibiting infection has been validated. See the “EXAMPLE” sectionbelow.

In one aspect, the present invention provides a method for selectingmonoclonal single chain antibody (scFv) against a peptide target. Asingle chain antibody generally includes a heavy chain variable region(V_(H)) of antibody covalently linked to a light chain variable region(V_(L)) of antibody via a peptide linker. In one embodiment, the methodcomprising:

expressing a library of scFv fusion proteins in yeast cells, each scFvfusion protein comprising either an activation domain or a DNA bindingdomain of a transcription activator and a scFv, the scFv comprising aV_(H) of antibody whose sequence varies within the library, a V_(L) ofantibody whose sequence varies within the library independently of theV_(H), and a linker peptide which links the V_(H) and V_(L);

expressing a target fusion protein in the yeast cells expressing thescFv fusion proteins, the target fusion protein comprising either theDNA binding domain or the activation domain of the transcriptionactivator which is not comprised in the scFv fusion proteins, and atarget peptide having a length of 5-100 amino acid residues (aa); and

selecting those yeast cells in which a reporter gene is expressed, theexpression of the reporter gene being activated by a reconstitutedtranscriptional activator formed by binding of the scFv fusion proteinto the target fusion protein.

According to the embodiment, the diversity of the library scFv fusionproteins is preferably higher than 1×10⁴, more preferably higher than1×10⁶, and most preferably higher than 1×10⁷.

Also according to the embodiment, the length of the target peptide ispreferably 10-80 aa, more preferably 20-60 aa, and most preferably 30-50aa.

Also according to the embodiment, the target peptide is preferably afragment of a membrane protein, more preferably an extracellular domainof a membrane protein, and most preferably an extracellular loop of atransmembrane protein.

Examples of a membrane protein include, but are not limited to,receptors for growth factors (e.g., vascular endothelial growth factor(VEGF), transforming growth factor (TGF), fibroblast growth factor (FF),platelet derived growth factor (PDGF), insulin-like growth factor),insulin receptor, insulin receptor, MHC proteins (e.g. class I MHC andclass II MHC protein), CD3 receptor, T cell receptors, cytokinereceptors such as interleukin-2 (IL-2) receptor,tyrosine-kinase-associated receptors such as Src, Yes, Fgr, Lck, Lyn,Hck, and Blk, and G-protein coupled receptors such as G-protein coupledreceptors such as receptors for the hormone relaxin (LGR7 and LGR8) andcoreceptors for HIV (e.g., CXCR4, CCR5, CCR1, CCR2b, CCR3, CCR4, CCR8,CXCR1, CXCR2, CXCR3, CX₃CR1, STRL33/BONZO and GPR15/BOB).

By using the method of present invention, antibodies with high affinityand specificity can be selected by screening a library of scFvantibodies against the target peptide expressed as a fusion protein inyeast. Compared to conventional approaches of generating monoclonalantibody by hybridoma technology and the recently developed XENOMOUSE®technology, the present invention provides a more efficient andeconomical way to screen for fully human antibodies against virtuallyany target peptide in a much shorter period of time. More importantly,the screening of the antibody libraries can be readily adopted for highthroughput screening in vivo.

In particular, the method of the present invention has been used forscreening fully human antibody library against HIV coreceptors such asCCR5 and CXCR4 in yeast. Significantly, single chain antibodies againstfragments of CCR5 have been selected and demonstrated to bind to humanCCR5 with high affinity and inhibit HIV-1 infection at sub-nanomolarconcentrations.

An advantage of the present invention is that the overall process ofscreening is very efficient and high throughput. For any targetedmembrane protein, each domain (or fragment) of the protein can be cansystematically screened against the same library of human antibody withhigh diversity (>1×10⁷). Since the peptide comprising the domain isexpressed intracellularly and screened for binding with the library ofantibody intracellulary, the peptide needs not be isolated orsynthesized in vitro, thus greatly simplifying the process and reducinglabor and cost.

Further, the fast proliferation rate of yeast cells and ease of handlingmakes a process of “molecular evolution” dramatically shorter than thenatural process of antibody affinity maturation in a mammal. Therefore,antibody repertoires with extremely high diversity can be produced andscreened directly against the fusion protein containing the targetpeptide in yeast cells at a much lower cost and higher efficiency thanprior processes such as the painstaking, stepwise “humanization” ofmonoclonal murine antibodies isolated by using the conventionalhybridoma technology (a “protein redesign”) or the recently-developedXENOMOUSE™ technology.

According to the “protein redesign” approach, murine monoclonalantibodies of desired antigen specificity are modified or “humanized” invitro in an attempt to reshape the murine antibody to resemble moreclosely its human counterpart while retaining the originalantigen-binding specificity. Riechmann et al. (1988) Nature 332:323-327.This humanization demands extensive, systematic genetic engineering ofthe murine antibody, which could take months, if not years.Additionally, extensive modification of the backbone of the murinemonoclonal antibody may result in reduced specificity and affinity.

In comparison, by using the method of the present invention, fully humanantibodies with high affinity to a specified target peptide can bescreened and isolated directly from yeast cells without going throughsite-by-site modification of the antibody, and without sacrifice ofspecificity and affinity of the selected antibodies.

The XENOMOUSE™ technology has been used to generate fully humanantibodies with high affinity by creating strains of transgenic micethat produce human antibodies while suppressing the endogenous murine Igheavy- and light-chain loci. However, the breeding of such strains oftransgenic mice and selection of high affinity antibodies can take along period of time. The antigen against which the pool of the humanantibody is selected has to be recognized by the mouse as a foreignantigen in order to mount immune response; antibodies against a targetantigen that does not have immunogenicity in a mouse may not be able tobe selected by using this technology.

In contrast, by using the method of the present invention, any peptidefragment derived the target protein can be expressed as a fusion proteinwith a DNA-binding domain (or an activation domain) of a transcriptionactivator and selected against the library of antibody in ayeast-2-hybrid system. Moreover, multiple peptide targets may be arrayedin multiple-well plates and screened against the library of antibodiesin a high throughput and automated manner.

Also compared to other approaches using transgenic goats and chickens toproduce antibodies, the method of the present invention can be used toscreen and produce fully human antibodies in large amounts withoutinvolving serious regulatory issues regarding the use of transgenicanimals, as well as safety issues concerning containment of transgenicanimals infected with recombinant viral vectors.

Various aspects of the present invention are described in detail in thefollowing sections.

1. Peptide Fragment from a Membrane Protein as the Target Peptide

In a preferred embodiment, the target peptide is a fragment of amembrane protein. The target peptide is expressed in yeast as a targetfusion protein with either a DNA binding domain or an activation domainof a transcription activator which is not comprised in the scFv fusionproteins. The epitope on the target peptide is presented by the targetfusion protein in yeast and recognized by some member(s) in the libraryof scFv fusion proteins. Such interactions trigger expression of areporter gene within the same cell, allowing identification of the yeastclones expressing the binding scFv fusion protein.

Member protein is a protein that is associated with the plasma membraneof a cell. Plasma membrane encloses the cell by forming a selectivepermeability barrier, defines its boundaries, and maintains theessential differences between the cytosol and the extracellularenvironmen. The plasma membrane consists lipids, proteins, and somecarbohydrates. Lipids form a bilayer in which the membrane proteins areembedded to varying degrees.

Different membrane proteins are associated with the membranes indifferent ways. Many membrane proteins extend through the lipid bilayer,with part of their mass on either side. These transmembrane proteins areamphipathic, having regions that are hydrophobic and regions that arehydrophilic. Their hydrophilic regions are exposed to water on one orthe other side of the membrane. Other membrane proteins are locatedentirely in the cytosol and are associated with the bilayer only bymeans of one or more covalently attached fatty acid chains or othertypes of lipid chains called prenyl groups. Yet other membrane proteinsare entirely exposed at the external cell surface, being attached to thebilayer only the covalent linkage (e.g., via a specific oligosaccharide)to phosphatidylinositol in the outer lipid monolayer of the plasmamembrane.

In a more preferred embodiment, the membrane protein is a transmembraneprotein. Typically, a transmembrane protein has its cytoplasmic andextracellular domains which are separated by the membrane-spanningsegments of the polypeptide chain. The membrane-spanning segmentscontact the hydrophobic environment of the lipid bilayer and arecomposed largely of amino acid residues with non-polar side chains. Thegreat majority of transmembrane proteins are glycosylated. Theoligosaccharide chains are usually present in the excellular domain.Further, the reducing environment of the cytosol prevents the formationof intrachain (and interchain) disulfide (S—S) bonds between cysteineresidues on the cytosolic side membranes. These disulfide bonds do formon the extracellular side, e.g., between the N-terminal domain and anextracellular loop.

Transmembrane proteins are notoriously difficult to crystallize forX-ray structural studies. The folded three-dimensional structures arequite uncertain for the isolated forms of these proteins. Thus, thesefeatures present a problem in the attempt to use the whole transmembraneprotein as a target for isolating molecules that would bind to it invitro.

According to the present invention, a peptide fragment derived from oneof the extracellular domains of the transmembrane protein could serve asthe target peptide. Antibody selected by using the screening method ofpresent invention binds to the exacelluar cellular domain, therebyeffectively blocking interactions of the transmembrane protein with itsexcellular ligand.

A family of transmembrane proteins called G protein-coupled receptors(GPCR) play important roles in the signal transduction process of acell. GPCR mediates the cellular responses to an enormous diversity ofsignaling molecules, including hormones, neurotransmitters, and localmediators. The signal molecules vary in their structure and function,including proteins, small peptides, as well as amino acid and fatty acidderivatives.

For example, receptors for the hormone relaxin (LGR7 and LGR8) have beenfound recently to be G-protein coupled receptors. Hsu et al. (2002)Science 295:671-674. Relaxin is a hormone important for the growth andremodeling of reproductive and other tissues during pregnancy. Hsu et aldemonstrated that two orphan heterotrimeric guanine nucleotide bindingprotein (G-protein) receptors, LGR7 and LGR8 are capable of mediatingthe action of relaxin through an adenosine 3′,5′-monophosphate(cAMP)-dependent pathway distinct from that of the structurally relatedinsulin and insulin-like growth factor. These receptors for relaxin areimplicated to play roles in reproductive, brain, renal, cardiovascularand other functions.

Despite the chemical and functional diversity of the signaling moleculesthat bind to them, all of GPCRs share a structural similarity in thatthe polypeptide chain threads back and forth across the lipid bilayerseven times, forming 7 transmembrane domains which are connected by 3extracellular loops and 3 intracellular loops.

Both CCR5 and CXCR4 are chemokine receptors are members of the GPCRsuperfamily. FIG. 1A shows the amino acid sequence of human CCR5 with 7transmembrane domains that are connected by loops 2, 4, and 6 which areextracellular loops and by loops 1, 3, 5 which are intracellular loops.FIG. 1B shows a model of the secondary structure of human CCR5. Blanpainet al. (1999) J. Biol. Chem. 274:34719-34727.

In particular, peptides derived from excellular loops of the membraneprotein could serve an ideal target for screening against the library ofantibody.

Other than CCR5 and CXCR4, examples of a chemokine receptor or achemokine receptor-like orphan receptor also include, but are notlimited to, CCR1, CCR2b, CCR3, CCR4, CCR8, CXCR1, CXCR2, CXCR3, CX₃CR1,STRL33/BONZO and GPR15/BOB. Berger, E. a. (1997) AIDS 11, Suppl. a:S3-S16; and Dimitrov, D. S. (1997) Cell 91: 721-730. Each or a set ofthese HIV coreceptors can mediate entry of different strains of HIVvirus into the host cell.

By using the method of the present invention, high affinity monoclonalantibodies can be generated against a peptide fragment of a chemokinereceptor efficiently and in a high throughput manner. Administering oneor more of these antibodies to a host may offer protection against orinhibit infection of HIV strains with broad-spectrum tropisms.

Other membrane proteins described above, the target peptide may bederived from any protein. For example, the target peptide may be derivedfrom a disease-associated antigen, such as tumor surface antigen such asB-cell idiotypes, CD20 on malignant B cells, CD33 on leukemic blasts,and HER2/neu on breast cancer. Antibody selected against these antigenscan be used in a wide variety of therapeutic and diagnosticapplications, such as treatment of cancer by direct administration ofthe antibody itself or the antibody conjugated with a radioisotope orcytotoxic drug, and in a combination therapy involving coadministrationof the antibody with a chemotherapeutic agent, or in conjunction withradiation therapy.

Alternatively, the target peptide may be derived from a growth factorreceptor. Examples of the growth factor include, but are not limited to,epidermal growth factors (EGFs), transferrin, insulin-like growthfactor, transforming growth factors (TGFs), interleukin-1, andinterleukin-2. For example, high expression of EGF receptors have beenfound in a wide variety of human epithelial primary tumors. TGF-A havebeen found to mediate an autocrine stimulation pathway in cancer cells.Several murine monoclonal antibody have been demonstrated to be able tobind EGF receptors, block the binding of ligand to EGF receptors, andinhibit proliferation of a variety of human cancer cell lines in cultureand in xenograft medels. Mendelsohn and Baselga (1995) “Antibodies togrowth factors and receptors”, in Biologic Therapy of Cancer, 2^(nd)Ed., J B Lippincott, Philadelphia, pp607-623; Leget and Czuczman (1998)“Use of rituximab, the new FDA-approved antibody’. Curr Opin Oncol.10:548-551; and Goldenberg (1999) “Trastuzumab, a recombinantDNA-derived humanized monoclonal antibody, a novel agent for thetreatment of metastatic breast cancer. Clin Ther. 21:309-318). Thus,fully human antibodies selected against these growth factors by usingthe method of the present invention can be used to treat a variety ofcancer.

The target peptide may also be derived from a cell surface protein orreceptor associated with coronary artery disease such as plateletglycoprotein Iib/IIIa receptor, autoimmune diseases such as CD4,CAMPATH-1 and lipid A region of the gram-negative bacteriallipopolysaccharide. Humanized antibodies against CD4 has been tested inclinical trials in the treatment of patients with mycosis fungoides,generalized postular psoriasis, severe psorisis, and rheumatoidarthritis. Antibodies against lipid A region of the gram-negativebacterial lipopolysaccharide have been tested clinically in thetreatment of septic shock. Antibodies against CAMPATH-1 has also beentested clinically in the treatment of against refractory rheumatoidarthritis. Thus, fully human antibodies selected against these growthfactors by using the method of the present invention can be used totreat a variety of autoimmune diseases (Vaswani et al. (1998) “Humanizedantibodies as potential therapeutic drugs” Annals of Allergy, Asthma andImmunology 81:105-115); inflammation (Present et al. (1999) “Infliximabfor the treatment of fistulas in patients with Crohn's disease” N Engl JMed. 340:1398-1405); and immuno-rejection in transplantation (Nashan etal. (1999) “Reduction of acute renal allograft rejection by daclizumab.Daclizumab Double Therapy Study Group”, Transplantation 67:110-115.

The target peptide may also be derived from proteins associated withhuman allergic diseases, such as those inflammatory mediator protein,e.g. Interleukin-1 (IL-1), tumor necrosis factor (TNF), leukotrienereceptor and 5-lipoxygenase, and adhesion molecules such as V-CAMNLA-4.In addition, IgE may also serve as the target antigen because IgE playspivotal role in type I immediate hypersensitive allergic reactions suchas asthma. Studies have shown that the level of total serum IgE tends tocorrelate with severity of diseases, especially in asthma. Burrows etal. (1989) “Association of asthma with serum IgE levels and skin-testreactivity to allergens” New Engl. L. Med. 320:271-277. Thus, fullyhuman antibodies selected against IgE by using the method of the presentinvention may be used to reduce the level of IgE or block the binding ofIgE to mast cells and basophils in the treatment of allergic diseaseswithout having substantial impact on normal immune functions.

The target peptide may also be derived from a viral surface or coreprotein which may serve as an antigen to trigger immune response of thehost. Examples of these viral proteins include, but are not limited to,glycoproteins (or surface antigens, e.g., GP120 and GP41) and capsidproteins (or structural proteins, e.g., P24 protein); surface antigensor core proteins of hepatitis A, B, C, D or E virus (e.g. smallhepatitis B surface antigen (SHBsAg) of hepatitis B virus and the coreproteins of hepatitis C virus, NS3, NS4 and NS5 antigens); glycoprotein(G-protein) or the fusion protein (F-protein) of respiratory syncytialvirus (RSV); surface and core proteins of herpes simplex virus HSV-1 andHSV-2 (e.g., glycoprotein D from HSV-2). For example, humanizedmonoclonal antibody has been developed for the prevention of respiratorysyncytial virus (RSV) infection. Storch (1998) “Humanized monoclonalantibody for prevention of respiratory syncytial virus infection”Pediatrics. 102:648-651.

The target peptide may also be derived from a mutated tumor suppressorgene that have lost its tumor-suppressing function and may render thecells more susceptible to cancer. Tumor suppressor genes are genes thatfunction to inhibit the cell growth and division cycles, thus preventingthe development of neoplasia. Mutions in tumor suppressor genes causethe cell to ignore one or more of the components of the network ofinhibitory signals, overcoming the cell cycle check points and resultingin a higher rate of controlled cell growth—cancer. Examples of the tumorsuppressor genes include, but are not limited to, DPC-4, NF-1, NF-2, RB,p53, WT1, BRCA1 and BRCA2.

DPC-4 is involved in pancreatic cancer and participates in a cytoplasmicpathway that inhibits cell division. NF-1 codes for a protein thatinhibits Ras, a cytoplasmic inhibitory protein. NF-1 is involved inneurofibroma and pheochromocytomas of the nervous system and myeloidleukemia. NF-2 encodes a nuclear protein that is involved in meningioma,schwanoma, and ependymoma of the nervous system. RB codes for the pRBprotein, a nuclear protein that is a major inhibitor of cell cycle. RBis involved in retinoblastoma as well as bone, bladder, small cell lungand breast cancer. P53 codes for p53 protein that regulates celldivision and can induce apoptosis. Mutation and/or inaction of p53 isfound in a wide ranges of cancers. WT1 is involved in Wilms tumor of thekidneys. BRCA1 is involved in breast and ovarian cancer, and BRCA2 isinvolved in breast cancer. Thus, fully human antibodies selected againsta mutated tumor suppressor gene product by using the method of thepresent invention can be used to block the interactions of the geneproduct with other proteins or biochemicals in the pathways of tumoronset and development.

2. Antibody Against Loop 6 of CCR5

The inventors also discovered that certain fragments derived from loop 6of CCR5 (designated hereafter “CCR5 Loop 6”) present excellent epitopesfor recognition by antibodies. The epitope(s) on CCR5 Loop 6 can be usedto elicit antibody by using the method of present invention or othermethods for generating antibody known in the art.

CCR5 Loop 6 includes amino acid residue aa 261-277: QEFFGLNNCSSSNRLDQ[SEQ ID NO:2] (shown in FIG. 1A). As demonstrated in the section of“EXAMPLE”, a peptide fragment containing most of the Loop 6 region and aportion of transmembrane domain 7, EFFGLNNCS SSNRLDQAMQ VTETLGMTHC [SEQID NO:3], could elicit monoclonal antibodies that bind to CCR5 with highaffinity and inhibit HIV-1 infection at sub-nanomolar concentrations.

According to the present invention, a peptide comprising a substantialportion of Loop 6 may serve as an epitope for elicit antibodies by usingthe method of the present invention or conventional methods such ashybridoma techniques and bacteriophage display panning. The antibodiesagainst CCR5 Loop 6 include but are not limited to polyclonal,monoclonal, Fab fragments, single chain antibodies, chimeric antibodies,etc.

For the production of antibodies against CCR5 Loop 6, various hostanimals may be immunized by injection with a peptide comprising aportion of CCR5 Loop 6. Such host 2 animals may include but are notlimited to rabbits, mice, and rats, to name but a few. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a peptide derived from CCR5 Loop 6, or an antigenic functionalderivative thereof. For the production of polyclonal antibodies, hostanimals such as those described above, may be immunized by injectionwith a peptide comprising a portion of CCR5 Loop 6 supplemented withadjuvants as also described above. It may be useful to conjugate thepeptide to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin (KLH), serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor, by using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydrid or SOCl₂.

Briefly, animals are immunized against CCR5 Loop 6 peptide or itsimmunogenic conjugates by combining, e.g., 100 μg or 5 μg of the proteinor conjugate (for rabbits or mice, respectively) with 3 volumes ofFreund's complete adjuvant and injecting the solution intradermally atmultiple sites. One month later the animals are boosted with ⅕ or 1/10the original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates can also be made in recombinant cell culture as proteinfusions. In addition, aggregating agents such as alum are suitably usedto enhance the immune response.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein (1975) Nature 256:495-497; and U.S. Pat. No.4,376,110, the human B-cell hybridoma technique (Kosbor et al. (1983)Immunology Today 4:72; Cole et al. (1983) Proc. Natl. Acad. Sci. USA80:2026-2030, and the EBV-hybridoma technique (Cole et al. (1985)Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo.

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster or macaque monkey, is immunized as herein above described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the antigen used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes arethen fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell. Goding (1986) “MonoclonalAntibodies: Principles and Practice”, pp. 59-103, Academic Press.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthaineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridoma typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred meyloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOP-21 and M.C.-11 mouse tumors, SP-2 or X63-Ag8-653 cellsavailable from the American Typeure Collection (ATCC), Rockville, Md.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies.

Culture medium in which hybridoma cell are growing is assayed forproduction of monoclonal antibodies directed against CCR5 Loop 6antigen. Preferably, the binding specificity of monoclonal antibodiesproduced by hybridoma cells is determined by immunoprecipitation or byan in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Suitable culture media for this purpose include, for example, DEME orRPMI-1640 medium. In addition, hybridoma cell may be grown in vivo asascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum, byconventional immunoglobulin purification procedures such as proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transferred into host cell suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells.

Monoclonal antibodies against CCR5 Loop 6 may also generated by usingbacteriophage display. Combinatorial libraries of antibodies have beengenerated in bacteriophage lambda expression systems which are screenedas bacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313). Also see review by Rader, C. and Barbas, C. F. (1997) “Phagedisplay of combinatorial antibody libraries” Curr. Opin. Biotechnol.8:503-508.

Various scFv libraries displayed on bacteriophage coat proteins havebeen described. Marks et al. (1992) Biotechnology 10: 779; Winter G andMilstein C (1991) Nature 349: 293; Clackson et al. (1991) op.cit.; Markset al. (1991) J. Mol. Biol. 222: 581; Chaudhary et al. (1990) Proc.Natl. Acad. Sci. (USA) 87: 1066; Chisweil et al. (1992) TIBTECH 10: 80;and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879.

Generally, a phage library is created by inserting a library of a randomoligonucleotide or a cDNA library encoding antibody fragment such asV_(L) and V_(H) into gene 3 of M13 or fd phage. Each inserted gene isexpressed at the N-terminal of the gene 3 product, a minor coat proteinof the phage. As a result, peptide libraries that contain diversepeptides can be constructed. The phage library is then affinity screenedagainst immobilized target molecule of interest, such as a peptidecomprising a portion of CCR5 Loop6, and specifically bound phages arerecovered and amplified by infection into Escherichia coli host cells.Typically, the target molecule of interest, such as a peptide comprisinga portion of CCR5 Loop6, is immobilized by covalent linkage to achromatography resin to enrich for reactive phage by affinitychromatography) and/or labeled for screen plaques or colony lifts. Thisprocedure is called biopanning. Finally, amplified phages can besequenced for deduction of the specific antibody sequences.

In addition, techniques developed for the production of “chimericantibodies” or “humanized antibodies” may be utilized to modify mousemonoclonal antibodies to reduce immunogenicity of non-human antibodies.Morrison et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neubergeretal. (1984) Nature, 312:604-608; Takeda et al. (1985) Nature,314:452-454. Such antibodies are generated by splicing the genes from amouse antibody molecule of appropriate antigen specificity together withgenes from a human antibody molecule of appropriate biological activitycan be used. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird (1988) Science 242:423-426;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al. (1989) Nature 334:544-546) can be adapted to producedifferentially expressed or pathway gene-single chain antibodies. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.(1989) Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

By using the method of the present invention in a yeast two-hybridsystem, three monoclonal scFv antibodies were selected. FIG. 7 shows ahomology alignment of the amino acid sequences of the three scFvantibodies. As shown in FIG. 7, other than the framework regions, thereis also substantial homology between the three scFv antibodies in heavychain CDR2 (in sequence GSTX₁YNPSL [SEQ ID NO: 32], X₁=N or T) and lightchain CDR2 (DAX₂X₃L [SEQ ID NO: 33], X₂=T or S, and X₃=T or D) regions.Thus, mutants of the three antibodies may be generated while conservingthe consensus sequences in the heavy and/or light chain CDR2 regions.

In one embodiment, an antibody is provided that binds to loop 6 of humanCCR5. In a variation, the antibody is capable of inhibiting HIV-1infection of human cells.

Optionally, CDR2 of the heavy chain variable region of the antibodycomprises amino acid sequence GSTX₁YNPSL [SEQ ID NO: 32], wherein X₁ isasparagine (N) or threonine (T).

Optionally, CDR2 of the light chain variable region comprises amino acidsequence DAX₂X₃L [SEQ ID NO: 33], wherein X₂ is threonine (T) or serine(S), and X₃ is threonine (T) or aspartic acid (D).

Optionally, CDR2 of the heavy chain variable region of the antibodycomprises amino acid sequence GSTX₁YNPSL [SEQ ID NO: 32]; and CDR2 ofthe light chain variable region comprises amino acid sequence DAX₂X₃L[SEQ ID NO: 33], wherein X₁ is asparagine (N) or threonine (T), X₂ isthreonine (T) or serine (S), and X₃ is threonine (T) or aspartic acid(D).

Optionally, CDR3 of the heavy chain variable region of the monoclonalantibody comprises 5, 6, 7, 8, 9 or more consecutive amino acids of asequence elected from the group consisting of

RLKGAWLLSEPPYFSSDGMDV [SEQ ID NO: 43],

RTVAGTSDY [SEQ ID NO: 44], and

HEQYYYDTSGQPYYFDF [SEQ ID NO: 45].

Optionally, CDR3 of the light chain variable region of the monoclonalantibody comprises 5, 6, 7, 8, 9 or more consecutive amino acids of asequence elected from the group consisting of

AAWDESLNGVV [SEQ ID NO: 46],

LQHDNFPLT [SEQ ID NO: 47], and

QQSDYLPLT [SEQ ID NO: 48].

Optionally, CDR3 of the heavy chain variable region of the monoclonalantibody comprises an amino acid sequence selected from the groupconsisting of SEQ ID Nos: 43-45; and CDR3 of the light chain variableregion of the monoclonal antibody comprises an amino acid sequenceselected from the group consisting of SEQ ID Nos: 46-48.

It is noted that the above-described different CDR regions may all beincluded in the antibody independent of each other, or in combinationwith one or more of each other.

Optionally, the heavy chain variable region of the monoclonal antibodycomprises an amino acid sequence selected from SEQ ID Nos: 36, 38, and40 (shown in FIG. 8).

Optionally, the light chain variable region of the monoclonal antibodycomprises an amino acid sequence selected from SEQ ID Nos: 37, 39, and41 (shown in FIG. 8).

It should be appreciated that the present invention also provides foranalogs of antibodies against CCR5 Loop 6 obtained according to themethods described above. Analogs may differ from naturally occurringproteins by conservative amino acid sequence differences or bymodifications which do not affect sequence, or by both.

For example, conservative amino acid changes may be made, which althoughthey alter the primary sequence of the peptide, do not normally alterits function. Conservative amino acid substitutions typically includesubstitutions within the following groups:

glycine, alanine;

valine, isoleucine, leucine;

aspartic acid, glutamic acid;

asparagine, glutamine;

serine, threonine;

lysine, arginine;

phenylalanine, tyrosine.

Modifications of the antibodies include in vivo, or in vitro chemicalderivatization of proteins, e.g., acetylation, or carboxylation. Alsoincluded are modifications of glycosylation, e.g., those made bymodifying the glycosylation patterns of a protein during its synthesisand processing or in further processing steps; e.g., by exposing theprotein to enzymes which affect glycosylation, e.g., mammalianglycosylating or deglycosylating enzymes. Also embraced are sequenceswhich have phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

Also included are antibodies which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties. Analogs ofsuch proteins include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The proteins of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

3. Use of Antibody Against HIV Coreceptors for Prevention and Treatmentof HIV Infection

The antibodies of the present invention selected against target peptidesderived from HIV coreceptors may be used for prevention and treatment ofHIV infection in vitro and in vivo.

To inhibit infection of cells by HIV in vitro, cells are treated withthe antibody of the invention, or a derivative thereof, either prior toor concurrently with the addition of virus. Inhibition of infection ofthe cells by the antibody of the present invention is assessed bymeasuring the replication of virus in the cells, by identifying thepresence of viral nucleic acids and/or proteins in the cells, forexample, by performing PCR, Southern, Northern or Western blottinganalyses, reverse transcriptase (RT) assays, or by immunofluorescence orother viral protein detection procedures. The amount of antibody andvirus to be added to the cells will be apparent to one skilled in theart from the teaching provided herein.

To prevent or inhibit infection of cells by HIV in vivo, the antibody ofthe present invention, or a derivative thereof, is administered to ahuman subject who is either at risk of acquiring HIV infection, or whois already infected with HIV.

The antibody of the present invention may be formulated for delivery viavarious routes of administration, including but not limited to, orally,parenterally, intraperitoneally, intravenously, intraarterially,topically, transdermally, sublingually, intramuscularly, rectally,transbuccally, intranasally, liposomally, via inhalation, vaginally,intraoccularly, via local delivery (for example by catheter or stent),subcutaneously, intraadiposally, intraarticularly, or intrathecally.

In an embodiment, the antibody is in an injectable formulation. Theformulation is suitable for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampules or inmultidose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Prior to administration, the antibody, or a derivative thereof, issuspended in a pharmaceutically acceptable formulation such as a salinesolution or other physiologically acceptable solution which is suitablefor the chosen route of administration and which will be readilyapparent to those skilled in the art of antibody preparation andadministration. The dose of antibody to be used may vary dependent uponany number of factors including the age of the individual, the route ofadministration and the extent of HIV infection in the individual. Theantibody is prepared for administration by being suspended or dissolvedin a pharmaceutically acceptable carrier such as saline, salt solutionor other formulations apparent to those skilled in such administration.

Typically, the antibody is administered in a range of 0.1 μg to 1 g ofprotein per dose. Approximately 1-10 doses are administered to theindividual at intervals ranging from once per day to once every fewyears.

The antibody may optionally be administered orally to a human. Forexample, the antibody of the present invention would be formulated inpropylene glycol solution by attaching the antibody a polymer carrier.Polymers or liposomes can stabilize the protein and desensitize it todigestive enzymes by encapsulating the protein within.

Also optionally, the antibody may be formulated for pulmonary deliveryvia inhalation. For example, the antibody could be delivered asaerosolized powder to a host using an inhaler. The lung provides anexcellent site for delivery of protein or peptide drug because the drugsare absorbed quickly into the blood-stream due to the huge surface areaof the lung. In addition, the layer separating airflow from bloodvessels is very narrow, so that the drug does not have far to travel toenter blood.

Also optionally, the antibody of the invention may be administered to ahost in a sustained release formulation using a biodegradablebiocompatible polymer, or by on-site delivery using micelles, gels andliposomes, or rectally (e.g., by suppository or enema). For example, theantibody is formulated with a polymer such as pluronic F127. The gelformulation may be injected subcutaneously or intramuscularly to allowthe antibody to be bled out over a period of time to ensure efficacy.

Also optionally, the antibody of the invention may be administered to ahost in a topical formulation. The antibody may be formulated withsuitable pharmaceutically acceptable carrier that does not denature orinactivate the protein in the form of lotion, cream, gel or suppository.For example, the anti-human CCR5 antibody of the present invention maybe used as prophylactic or therapeutic to prevent or treat infection ofHIV (or other sexually transmitted diseases or STD) via skin or mucosaof the body. The topical formulation of the antibody may be applied toall areas of skin likely to come in intimate contact during sexualactivity, especially to any area that has sores or breaks in the skin.For example, cream or lotion containing the antibody may be applied tothe surfaces of the penis, the base of the penis and scrotum, the uppervagina, the inner and outer lips of the vulva, the inner thighs, pubicand perianal regions. The antibody may also be applied to the anusand/or delivered directly to the rectum via the penis. In addition, theantibody may be incorporated into an intrauterine device or anintravaginal device that timely releases the antibody into the uterus orinto the vagina to provide continuous protection against infection ofviruses. For example, the antibody may be formulated as co-polymer withethylene-vinyl acetate which forms a soft, rubber-like material. Theprocedures for forming an antibody co-polymer with ethylene-vinylacetate are described in U.S. Pat. No. 4,391,797 which is incorporatedherein by reference in its entirety.

Applying the antibody to the skin and mucosa of the body is advantageousin that the surfaces of skin and mucus epithelia that are exposed tosemen and other body fluids during sexual activity are most at risk ofexposure to HIV or other STD pathogens. It is believed that the majorroles of secreted antibodies are to block the adhesive groups thatenable a pathogen to adhere to its target cell. The antibody of thepresent invention can be used to block the adhesion of HIV to its targetcells such as CD4⁺ cells by binding to HIV coreceptor such as CCR5 andCXCR4. With the occupation of the antibody on the coreceptors on thehost's cells, HIV carried by body fluid such as semen and blood ofanother individual can be prevented from entry into the host's cells,thus significantly reducing the risk of infection.

The antibody of the present invention may be used in combination with avariety of anti-retroviral drugs for prevention or treatment of HIVinfection. Anti-retroviral drugs include many small molecule drugs (e.g.organic compounds) and macromolecule drugs (antisense DNAs/RNAs,ribozymes, viral surface protein-binding proteins or nucleotides, etc.).

Anti-retroviral drugs against HIV have been developed since thediscovery of correlation between HIV and AIDS. In particular, manyanti-retroviral drugs have been developed to target critical enzymes ofretroviruses and inhibit replication of the virus inside the host cell.For example, nucleoside or nucleotide analogs such as AZT,dideoxycytidine (ddC), and dideoxyinosine (ddI) were developed toinhibit reverse transcriptase (RT) of retroviruses by acting ascompetitive inhibitors and chain terminators. Non-nucleoside ornucleotide inhibitors have also been found to inhibit reversetranscriptase activity of retroviruses by exerting an allosteric effectby binding to a hydrophobic pocket close to the active site of RT. Theprotease (PRO) inhibitors in current use are targeted at the active siteof the enzyme.

In addition to the RT and PRO inhibitors of HIV infection, other classesof antiviral agents targeting different components of HIV or interferingwith different stages of HIV life cycle may be also be used inconjunction with the antibody to achieve efficacious clinical results.For example, synthetic peptides have been modeled to mimic thecoiled-coiled helical bundle formed by heptad repeat sequences of one ofthe two subunits of HIV envelop glycoprotein, the transmembraneglycoprotein (gp41). Wild C. T. et al. “A synthetic peptide inhibitor ofHIV replication: correlation between solution structure and viralinhibition” Proc. Nat[. Acad. Sci. USA 89: 10537-10541 (1992). Theseheptad sequences play important roles in the conformational changesessential for membrane fusion of HIV with host cells. The syntheticpeptides, DP107 and DP178, have been shown to inhibit infection in vitroby disrupting the gp41 conformational changes associated with membranefusion. Wild, C. et al. “Peptides corresponding to a predictivealpha-helical domain of HIV-1 gp41 are potent inhibitors of virusinfection” Proc. Natl. Acad. Sci. USA 91: 9770-9774 (1994). Inparticular, a 36-amino acid peptide (T-20), corresponding to DP178,functions as a potent inhibitor of the HIV-1 envelop-cell membranefusion and viral entry. Wild, C. et al. “A synthetic peptide from HIV-1gp41 is a potent inhibitor of virus-mediated cell-cell fusion” AIDS Res.Hum. Retroviruses 9:1051-1053 (1993). When used in monotherapy, T-20demonstrated potent antiviral activity in vivo when administered as anintravenous subcutaneous infusion in trials of 28 days or less.Lalezari, J. et al “Safety, pharmacokinetics, and antiviral activity ofT-20 as a single agent in heavily pretreated patients” 6^(th) Conferenceon Retroviruses and Opportunistic Infections, Chicago, February 1999[Abstract LB13]. Such inhibitors of HIV fusion and entry into the hostcells may be combined with the antibodies of the present invention, aswell as other anti-retroviral agents to inhibit HIV infection atdifferent stages of the retroviral life cycle.

Further, inhibitors of retroviral integrase may be used in conjunctionwith in combination with the antibodies of the present inventionaccording to the present invention. A variety of inhibitors of HIVintegrase have been identified that inhibit HIV integration at differentstages. In general, retroviral integration occurs in the following threebiochemical stages: 1) assembly of a stable complex with specific DNAsequences at the end of the HIV-1 long terminal repeat (LTR) regions,(2) endonucleolytic processing of the viral DNA to remove the terminaldinucleotide from each 3′ end, and (3) strand transfer in which theviral DNA 3′ ends are covalently linked to the cellular (target) DNA.Pommier, Y. and Neamati, N. in Advances in Viral Research, K.Maramorosch, et al. eds. Academic Press, New York (1999), pp 427-458.Compounds have been identified to interfere with assembly of the stablecomplex in assays with purified, recombinant integrase. Hazuda, D. J. etal. Drug Des. Discovery 15: 17 (1997). In a random screening of morethan 250,000 samples. A variety of compounds have been discovered asinhibitors of strand transfer reaction catalyzed by integrase. Hazuda,D. J. et al. “Inhibitors of strand transfer that prevent integration andinhibit HIV-1 replication in cells” Science 287:646-650 (2000). The mostpotent and specific compounds each contained a distinct diketo acidmoiety, such as compound L-731,988, L-708,906, L-731,927, and L-731,942.Hazuda, D. J. et al. (2000), supra. Such inhibitors of HIV integrationinto the host genome may be combined with in combination with theantibodies of the present invention, as well as other anti-retroviralagents to inhibit HIV infection at different stages of the retrovirallife cycle.

In the pharmaceutical compositions of the present invention, nucleosidereverse transcriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, protease inhibitors, fusion inhibitors and integraseinhibitors are the preferred anti-retroviral drugs in combination withthe antibody. Examples of the nucleoside HIV reverse transcriptaseinhibitor include, but are not limited to zidovudine (AZT), didanosine(ddI), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), abacavir(1592U89), and adefovir dipivoxil (bis(POM)-PMEA). Examples of thenon-nucleoside HIV reverse transcriptase inhibitor include, but are notlimited to nevirapine (BI-RG-587), delavirdine (BHAP, U-90152) andefavirenz (DMP 266). Examples of the HIV protease inhibitors include,but are not limited to indinavir (MK-639), ritonavir (ABT-538),saqinavir (Ro-31-8959), nelfinavir (AG-1343), and amprenavir (141W94).

The antibody of the present invention may be used in combination withany one or more of the antiretroviral drugs, preferably with a“cocktail” of nucleoside reverse transcriptase inhibitors,non-nucleoside HIV reverse transcriptase inhibitors, and proteaseinhibitors. For example, the antibody of the present invention may becombined with two nucleoside reverse transcriptase inhibitors (e.g.zidovudine (AZT) and lamivudine (3TC)), and one protease inhibitor (e.g.indinavir (MK-639)). The antibody of the present invention may also becombined with one nucleoside reverse transcriptase inhibitor (e.g.stavudine (d4T)), one non-nucleoside reverse transcriptase inhibitor(e.g. nevirapine (BI-RG-587)), and one protease inhibitor (e.g.nelfinavir (AG-1343)). Alternatively, the antibody of the presentinvention may be combined with one nucleoside reverse transcriptaseinhibitor (e.g. zidovudine (AZT)), and two protease inhibitors (e.g.nelfinavir (AG-1343) and saqinavir (Ro-31-8959)).

Optionally, the pharmaceutical composition of the present inventionfurther includes one or more general antiviral agents. Examples ofgeneral antiviral agents include, but are not limited to acyclovir,ganciclovir, trisodium phosphonoformate, novapren (Novaferon Labs, Inc.,Akron, Ohio), Peptide T Octapeptide Sequence (Peninsula Labs, Belmont,Calif.), ansamycin LM 427 (Adria Labortories, Dublin, Ohio), dextransulfate, virazole, ribavirin (Virateck/ICN, Costa Mesa, Calif.),α-interferon, and β-interferon. General antiviral agents can be used toprevent or inhibit opportunistic infections of other viruses.

4. Use of Antibody Against HIV Coreceptors for Screening Anti-HIV Agents

The antibody of the present invention may also be used in a method ofscreening agents for anti-HIV activity. A test agent (e.g., a compound)is first screened for the ability to bind to the antibody of theinvention. Compounds which bind to the antibody are likely to sharestructural and perhaps biological activities with the HIV coreceptor(e.g., CCR5) and thus, may serve as competitive inhibitors forinhibition of the interaction of HIV envelope protein with CD4 and/orCCR5 plus CD4. An antibody-binding compound is further tested forantiviral activity by treating cells with the compound either prior toor concurrently with the addition of virus to the cells. Alternatively,the virus and the compound may be mixed together prior to the additionof the mixture to the cells. The ability of the compound to affect virusinfection is assessed by measuring virus replication in the cells usingany one of the known techniques, such as a RT assay, immunofluorescenceassays and other assays known in the art useful for detection of viralproteins or nucleic acids in cells. Generation of newly replicated virusmay also be measured using known virus assays such as those which aredescribed herein.

The antibody of the present invention may also be used in competitionassays to screen for compounds that bind to the HIV coreceptor (e.g.,CCR5) and which therefore prevent binding of the antibody to thecoreceptor. Such compounds, once identified, may be examined further todetermine whether or not they prevent entry of virus into cells.Compounds which prevent entry of virus into cells are useful asanti-viral compounds.

Additional uses for the antibody of the present invention include theidentification of cells in the body which are potential targets forinfection by HIV. These cells express HIV coreceptor(s) and aretherefore capable of being infected by HIV. For example, cells which arepotential targets for HIV infection may be identified by virtue of thepresence of CCR5 on their surface. The antibody of the present inventionfacilitates identification of these cells as follows.

The antibody of the present invention is first combined with anidentifiable marker, such as an immunofluorescent or radioactive marker.Cells which are obtained from a human subject are then reacted with thetagged antibody. Binding of the antibody to cells is an indication thatsuch cells are potential targets for HIV infection. The identificationof cells which may be infected with HIV is important for the design oftherapies for the prevention of HIV infection. In the case ofindividuals who are infected with HIV, the identification of targetcells provides an immune profile of these individuals which providesuseful information regarding the progress of their infection.

In addition to the aforementioned uses for the monoclonal antibody ofthe present invention, the antibody may be useful for the detection ofCCR5 on a variety of cell types on which CCR5 may be expressed.

The monoclonal antibody of the present invention may be useful formonitoring CCR5 expression levels on a variety of cell types, whichexpression may be an indication of a disease state in a human,including, but not limited to HIV infection, atherosclerosis, and thelike.

5. Construction of scFv Library via Homologous Recombination in Yeast

The library of scFv proteins may be produced in vivo or in vitro byusing any methods known in the art. In a preferred embodiment, thelibrary of scFv proteins is constructed in yeast by exploiting theintrinsic property of yeast—homologous recombination at an extremelyhigh level of efficiency.

FIG. 3 shows a flow chart delineating a method for generating andscreening highly diverse libraries of single-chain human antibodies(scFv) in yeast. As illustrated in FIG. 3, a highly complex library ofscFv is constructed in yeast cells. In particular, cDNA libraries of theheavy and light chain variable regions (V_(H) and V_(L)) are transferredinto a yeast expression vector by direct homologous recombinationbetween the sequences encoding V_(H) and V_(L), and the yeast expressionvector containing homologous recombination sites. The resultingexpression vector is called scFv expression vector. This primaryantibody library may reach a diversity preferably between 10⁶-10¹², morepreferably between 10⁷-10¹², and most preferably between 10⁸-10¹².

The diversity of V_(H) and V_(L) within the library of scFv fusionproteins may be preferably between 10³-10⁸, more preferably between10⁴-10⁸, and most preferably between 10⁵-10⁸.

Optionally, AD is an activation domain of yeast GAL 4 transcriptionactivator; and BD is a DNA binding domain of yeast GAL 4 transcriptionactivator.

The linker sequence L may have a specific sequence, or vary within thelibrary of the yeast expression vectors.

The linker sequences L in the library of expression vectors ispreferably between 30-120 bp in length, more preferably between 45-102bp in length, and most preferably between 45-63 bp in length. The linkersequence in the library of expression vectors preferably comprises anucleotide sequence encoding an amino acid sequence ofGly-Gly-Gly-Gly-Ser in 3 or 4 tandem repeats.

The linker peptides expressed by the library of expression vectorspreferably provide a substantially conserved conformation between thefirst and second polypeptide subunits across the fusion proteinsexpressed by the library of expression vectors. For example, a linkerpeptide Gly-Gly-Gly-Gly-Ser [SEQ ID NO: 42] in 4 tandem repeats (G₄S)₄[SEQ ID NO: 4] is believed to provide a substantially conservedconformation of scFv antibodies which preserves its antigen-binding sitein the variable regions of the corresponding full antibody.

DNA sequences encoding human antibody V_(H) and V_(L) segments may bepolynucleotide segments of at least 30 contiguous base pairssubstantially encoding genes of the immunoglobulin superfamily. A. F.Williams and A. N. Barclay (1989) “The Immunoglobulin Gene Superfamily”,in Immunoglobulin Genes, T. Honjo, F. W. Alt, and T. H. Rabbitts, eds.,Academic Press: San Diego, Calif., pp.361-387. The V_(H) and V_(L) genesare most frequently encoded by human, non-human primate, avian, porcine,bovine, ovine, goat, or rodent heavy chain and light chain genesequences.

The library of DNA sequences encoding human antibody V_(H) and V_(L)segments may be derived from a variety of sources. For example, mRNAencoding the human antibody V_(H) and V_(L) libraries may be extractedfrom cells or organs from immunized or non-immunized animals or humans.Preferably, organs such as human fetal spleen and lymph nodes may beused. Peripheral blood cells from non-immunized humans may also be used.The blood samples may be from an individual donor, from multiple donors,or from combined blood sources.

The human antibody V_(H)- and V_(L)-coding sequences may be derived andamplified by using sets of oligonucleotide primers to amplify the cDNAof human heavy and light chains variable domains by polymerase chainreaction (PCR). Orlandi et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837. For example, blood sample may be from healthy volunteers andB-lymphocyte in the blood can be isolated. RNA can be prepared byfollowing standard procedures. Cathala et al. (1983) DNA 3:329. The cDNAcan be made from the isolated RNA by using reverse transcriptase.

Alternatively, the V_(H)- and V_(L)-coding sequences may be derived froman artificially rearranged immunoglobulin gene or genes. For example,immunoglobulin genes may be rearranged by joining of germ line Vsegments in vitro to J segments, and, in the case of V_(H) domains, Dsegments. The joining of the V, J and D segments may be facilitated byusing PCR primers which have a region of random or specific sequence tointroduce artificial sequence or diversity into the products.

The fusion protein formed by linking V_(H) and V_(L) polypeptides isalso referred as a single-chain antibody, scFv. A typical scFv comprisesa V_(H) domain and a V_(L) domain in polypeptide linkage, generallylinked via a spacer/linker peptide L. The linker peptide sequence L mayencode an appropriately designed linker peptide, such as(Gly-Gly-Gly-Gly-Ser)₄ [SEQ. ID NO: 4] or equivalent linker peptide(s).The linker bridges the C-terminus of the first V region and N-terminusof the second, ordered as either V_(H)-L-V_(L) or V_(L)-L-V_(H).

A scFv may comprise additional amino acid sequences at the amino- and/orcarboxy-termini. For example, a single-chain antibody may comprise atether segment for linking to the constant regions of a complete or fullantibody. A functional single-chain antibody generally contains asufficient portion of an immunoglobulin superfamily gene product so asto retain the property of binding to a specific target molecule,typically a receptor or antigen (epitope).

In a preferred embodiment, the expression vector is based on a yeastplasmid, especially one from Saccharomyces cerevisiae. Aftertransformation of yeast cells, the exogenous DNA encoding scFv fusionproteins are uptaken by the cells and subsequently expressed by thetransformed cells.

More preferably, the expression vector may be a yeast-bacteria shuttlevector which can be propagated in either Escherichia coli or yeastStruhl, et al. (1979) Proc. Natl. Acad. Sci. 76:1035-1039. The inclusionof E. coli plasmid DNA sequences, such as pBR322, facilitates thequantitative preparation of vector DNA in E. coli, and thus theefficient transformation of yeast.

The types of yeast plasmid vector that may serve as the shuttle may be areplicating vector or an integrating vector. A replicating vector isyeast vector that is capable of mediating its own maintenance,independent of the chromosomal DNA of yeast, by virtue of the presenceof a functional origin of DNA replication. An integrating vector reliesupon recombination with the chromosomal DNA to facilitate replicationand thus the continued maintenance of the recombinant DNA in the hostcell. A replicating vector may be a 2μ-based plasmid vector in which theorigin of DNA replication is derived from the endogenous 2μ plasmid ofyeast. Alternatively, the replicating vector may be an autonomouslyreplicating (ARS) vector, in which the “apparent” origin of replicationis derived from the chromosomal DNA of yeast. Optionally, thereplicating vector may be a centromeric (CEN) plasmid which carries inaddition to one of the above origins of DNA replication a sequence ofyeast chromosomal DNA known to harbor a centromere.

The vectors may be transformed into yeast cells in a closed circularform or in a linear form. Transformation of yeast by integratingvectors, although with inheritable stability, may not be efficient whenthe vector is in in a close circular form (e.g. 1-10 transformants perug of DNA). Linearized vectors, with free ends located in DNA sequenceshomologous with yeast chromosomal DNA, transforms yeast with higherefficiency (100-1000 fold) and the transforming DNA is generally foundintegrated in sequences homologous to the site of cleavage. Thus, bycleaving the vector DNA with a suitable restriction endonuclease, it ispossible to increase the efficiency of transformation and target thesite of chromosomal integration. Integrative transformation may beapplicable to the genetic modification of brewing yeast, providing thatthe efficiency of transformation is sufficiently high and the target DNAsequence for integration is within a region that does not disrupt genesessential to the metabolism of the host cell.

ARS plasmids, which have a high copy number (approximately 20-50 copiesper cell) (Hyman et al., 1982), tend to be the most unstable, and arelost at a frequency greater than 10% per generation. However, thestability of ARS plasmids can be enhanced by the attachment of acentromere; centromeric plasmids are present at 1 or 2 copies per celland are lost at only approximately 1% per generation.

The expression vector of the present invention is preferably based onthe 2μ plasmid. The 2μ plasmid is known to be nuclear in cellularlocation, but is inherited in a non-Mendelian fashion. Cells that lostthe 2μ plasmid have been shown to arise from haploid yeast populationshaving an average copy number of 50 copies of the 2μ plasmid per cell ata rate of between 0.001% and 0.01% of the cells per generation. Futcher& Cox (1983) J. Bacteriol. 154:612. Analysis of different strains of S.cerevisiae has shown that the plasmid is present in most strains ofyeast including brewing yeast. The 2μ plasmid is ubiquitous andpossesses a high degree of inheritable stability in nature.

The 2μ plasmid harbors a unique bidirectional origin of DNA replicationwhich is an essential component of all 2μ-based vectors. The plasmidcontains four genes, REP1, REP2, REP3 and FLP which are required for thestable maintenance of high plasmid copy number per cell Jaysram et al.(1983) Cell 34:95. The REP1 and REP2 genes encode trans-acting proteinswhich are believed to function in concert by interacting with the REP3locus to ensure the stable partitioning of the plasmid at cell division.In this respect, the REP3 gene behaves as a cis acting locus whicheffects the stable segregation of the plasmid, and is phenotypicallyanalogous to a chromosomal centromere. An important feature of the 2μplasmid is the presence of two inverted DNA sequence repeats (each 559base-pairs in length) which separate the circular molecule into twounique regions. Intramolecular recombination between the inverted repeatsequences results in the inversion of one unique region relative to theother and the production in vivo of a mixed population of two structuralisomers of the plasmid, designated A and B. Recombination between thetwo inverted repeats is mediated by the protein product of a gene calledthe FLP gene, and the FLP protein is capable of mediating high frequencyrecombination within the inverted repeat region. This site specificrecombination event is believed to provide a mechanism which ensures theamplification of plasmid copy number. Murray et al. (1987) EMBO J.6:4205.

The expression vector may also contain an Escherichia coli origin ofreplication and E. coli antibiotic resistance genes for propagation andantibiotic selection in bacteria. Many E. coli origins are known,including ColE1, pMB1 and pBR322, The ColE origin of replication ispreferably used in this invention. Many E. coli drug resistance genesare known, including the ampicillin resistance gene, thechloramphenoicol resistance gene and the tetracycline resistance gene.In one particular embodiment, the ampicillin resistance gene is used inthe vector.

The transformants that carry the scFv librarymay be selected by usingvarious selection schemes. The selection is typically achieved byincorporating within the vector DNA a gene with a discernible phenotype.In the case of vectors used to transform laboratory yeast, prototrophicgenes, such as LEU2, URA3 or TRP1, are usually used to complementauxotrophic lesions in the host. However, in order to transform brewingyeast and other industrial yeasts, which are frequently polyploid and donot display auxotrophic requirements, it is necessary to utilize aselection system based upon a dominant selectable gene. In this respectreplicating transformants carrying 2μ-based plasmid vectors may beselected based on expression of marker genes which mediate resistanceto: antibiotics such as G418, hygromycin B and chloramphenicol, orotherwise toxic materials such as the herbicide sulfometuron methyl,compactin and copper.

6. Screening of scFv Library Against the Target Peptide in YeastTwo-Hybrid System

The present invention provides efficient methods for screening the scFvlibrary against any target peptide in a yeast two-hybrid system.

The two-hybrid system is a selection scheme designed to screen forpolypeptide sequences which bind to a predetermined polypeptide sequencepresent in a fusion protein. Chien et al. (1991) Proc. Natl. Acad. Sci.(USA) 88: 9578). This approach identifies protein-protein interactionsin vivo through reconstitution of a transcriptional activator. Fieldsand Song (1989) Nature 340: 245), the yeast Gal 4 transcription protein.The method is based on the properties of the yeast Gal 4 protein, whichconsists of separable domains responsible for DNA-binding andtranscriptional activation. Polynucleotides encoding two hybridproteins, one consisting of the yeast Gal4 DNA-binding domain (BD) fusedto a polypeptide sequence of a known protein and the other consisting ofthe Gal4 activation domain (AD) fused to a polypeptide sequence of asecond protein, are constructed and introduced into a yeast host cell.Intermolecular binding between the two fusion proteins reconstitutes theGal4 DNA-binding domain with the Gal4 activation domain, which leads tothe transcriptional activation of a reporter gene (e.g., lacZ, HIS3)which is operably linked to a Gal4 binding site.

Typically, the two-hybrid method is used to identify novel polypeptidesequences which interact with a known protein. Silver and Hunt (1993)Mol. Biol. Rep. 17: 155; Durfee et al. (1993) Genes Devel. 7; 555; Yanget al. (1992) Science 257: 680; Luban et al. (1993) Cell 73: 1067; Hardyet al. (1992) Genes Devel. 6; 801; Bartel et al. (1993) Biotechniques14: 920; and Vojtek et al. (1993) Cell 74: 205. The two-hybrid systemwas used to detect interactions between three specific single-chainvariable fragments (scFv) and a specific antigen. De Jaeger et al.(2000) FEBS Lett. 467:316-320. The two-hybrid system was also used toscreen against cell surface proteins or receptors such as receptors ofhematopoietic super family in yeast. Ozenberger, B. A., and Young, K. H.(1995) “Functional interaction of ligands and receptors of hematopoieticsuperfamily in yeast” Mol Endocrinol. 9:1321-1329.

Variations of the two-hybrid method have been used to identify mutationsof a known protein that affect its binding to a second known protein Liand Fields (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc. Natl. Acad.Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol. 13; 2899;and Madura et al. (1993) J. Biol. Chem. 268: 12046.

Two-hybrid systems have also been used to identify interactingstructural domains of two known proteins or domains responsible foroligomerization of a single protein. Bardwell et al. (1993) Med.Microbiol. 8: 1177; Chakraborty et al. (1992) J. Biol. Chem. 267: 17498;Staudinger et al. (1993) J. Biol. Chem. 268: 4608; and Milne G T; WeaverD T (1993) Genes Devel. 7; 1755; Iwabuchi et al. (1993) Oncogene 8;1693; Bogerd et al. (1993) J. Virol. 67: 5030).

Variations of two-hybrid systems have been used to study the in vivoactivity of a proteolytic enzyme. Dasmahapatra et al. (1992) Proc. Natl.Acad. Sci. (USA) 89: 4159. Alternatively, an E. coli/BCCP interactivescreening system was used to identify interacting protein sequences(i.e., protein sequences which heterodimerize or form higher orderheteromultimers). Germino et al. (1993) Proc. Natl. Acad. Sci. (U.S.A.)90: 933; and Guarente L (1993) Proc. Natl. Acad. Sci. (U.S.A.) 90: 1639.

Typically, selection of binding protein using a two-hybrid method reliesupon a positive association between two Gal4 fusion proteins, therebyreconstituting a functional Gal4 transcriptional activator which theninduces transcription of a reporter gene operably linked to a Gal4binding site. Transcription of the reporter gene produces a positivereadout, typically manifested either (1) as an enzyme activity (e.g.,β-galactosidase) that can be identified by a calorimetric enzyme assayor (2) as enhanced cell growth on a defined medium (e.g., HIS3 and Ade2). Thus, the method is suited for identifying a positive interaction ofpolypeptide sequences, such as antibody-antigen interactions.

False positives clones that indicate activation of the reporter geneirrespective of the specific interaction between the two hybridproteins, may arise in the two-hybrid screening. Various procedures havedeveloped to reduce and eliminate the false positive clones from thefinal positives. For example, 1) prescreening the clones that containsthe target vector and shows positive in the absence of the two-hybridpartner (Bartel, P. L., et al. (1993) “Elimination of false positivesthat arise in using the two-hybrid system” BioTechniques 14:920-924); 2)by using multiple reporters such as His3, β-galactosidase, and Ade2(James, P. et al. (1996) “Genomic libraries and a host strain designedfor highly efficient two-hybrid selection in yeast” Genetics144:1425-1436); 3) by using multiple reporters each of which is underdifferent GAL 4-responsive promoters such as those in yeast strain Y190where each of the His 3 and p-Gal reporters is under the control of adifferent promoter Gal 1 or Gal 10, but both response to Gal 4 signaling(Durfee, T., et al (1993) “The retinoblastoma protein associates withthe protein phosphatase type 1 catalytic subunit” Genes Devel.7:555-569); and 4) by post-screening assays such as testing isolateswith target consisting of GAL 4-BD alone.

In addition, the false positive clones may also be eliminated by usingunrelated targets to confirm specificity. This is a standard controlprocedure in the two-hybrid system which can be performed after thelibrary isolate is confirmed by the above-described 1)-4) procedures.Typically, the library clones are confirmed by co-transforming theinitially isolated library clones back into the yeast reporter strainwith one or more control targets unrelated to the target used in theoriginal screening. Selection is conducted to eliminate those libraryclones that show positive activation of the reporter gene and thusindicate non-specfic interactions with multiple, related proteins.

When the library of scFv fusion proteins are expressed by the expressionvector in yeast cells, such as cells from the Saccharomyces cerevisiaestrains, the scFv fusion protein undergoes a process of protein foldingto adopt one or more conformations. The peptide sequence encoded by thelinker sequence L also facilitates the folding by providing a flexiblehinge between the V_(H) and V_(L). The conformation(s) adopted by thescFv fusion protein may have suitable binding site(s) for a specifictarget peptide expressed as fusion protein with the domain BD of atranscription activator. The AD domain of the scFv fusion protein shouldbe able to activate transcription of gene(s) once the AD and BD domainsare reconstituted to form an active transcription activator in vitro orin vivo by a two-hybrid method.

In a preferred embodiment, the highly complex primary antibody librariesis screened against the peptide target, for example a 30 aa peptidederived from loop 6 of CCR5. This screening for antibody-antigeninteraction is conveniently carried out in yeast by using a yeasttwo-hybrid method. The library of scFv expression vectors are introducedinto yeast cells. Expression of the scFv antibody library in the yeastcells produces a library of scFv fusion proteins, each fusion proteincomprising a scFv and an activation domain (AD) of a transcriptionactivator. The yeast cells are also modified to express a recombinantfusion protein comprising a DNA-binding domain (BD) of the transcriptionactivator and the target peptide. The yeast cells are also modified toexpress a reporter gene whose expression is under the control of aspecific DNA binding site. Upon binding of the scFv antibody from thelibrary to the target antigen, the AD is brought into close proximity ofBD, thereby causing transcriptional activation of a reporter genedownstream from a specific DNA binding site to which the BD binds. It isnoted that the library of scFv expression vectors may contain the BDdomain while the modified yeast cells express a fusion proteincomprising the AD domain and the target peptide.

These scFv expression vectors may be introduced to yeast cells byco-transformation of diploid yeast cells or by direct mating between twostrains of haploid yeast cells. For example, the scFv expression vectorsand an expression vector containing the target peptide can be used toco-transform diploid yeast cells in a form of yeast plasmid orbacteria-yeast shuttle plasmid. Alternatively, two strains haploid yeastcells (e.g. α- and a-type strains of yeast), each containing the scFvexpression vector and the target peptide expression vector,respectively, are mated to produce a diploid yeast cell containing bothexpression vectors. Preferably, the haploid yeast strain containing thetarget peptide expression vector also contains the reporter genepositioned downstream of the specific DNA binding site.

The yeast clones containing scFv antibodies with binding affinity to thetarget peptide are selected based on phenotypes of the cells or otherselectable markers. The plasmids encoding these primary antibody leadscan be isolated and further characterized. The affinity and biologicalactivity of the primary antibody leads can be determined using assaysparticularly designed based on the specific target protein from whichthe target peptide is derived.

FIG. 2A illustrates a flow diagram of a preferred embodiment of theabove described method. As illustrated in FIG. 2A, the sequence librarycontaining scFv fused with an AD domain upstream is carried by a libraryof expression vectors, the AD-scFv vectors. The coding sequence of thetarget peptide (labeled as “Target”) is contained in another expressionvector and fused with a BD domain, forming the BD-Target vector.

The AD-scFv vector and the BD-Target vector may be co-transformed into ayeast cell by using method known in the art. Gietz, D. et al. (1992)“Improved method for high efficiency transformation of intact yeastcells” Nucleic Acids Res. 20:1425. The construct carrying the specificDNA binding site and the reporter gene (labeled as “Reporter”) may bestably integrated into the genome of the host cell or transientlytransformed into the host cell. Upon expression of the sequences in theexpression vectors, the library of scFv fusion proteins undergo proteinfolding in the host cell and adopt various conformations. Some of thescFv fusion proteins may bind to the Target protein expressed by theBD-Target vector in the host cell, thereby bringing the AD and BDdomains to a close proximity in the promoter region (i.e., the specificDNA binding site) of the reporter construct and thus reconstituting afunctional transcription activator composed of the AD and BD domains. Asa result, the AD activates the transcription of the reporter genedownstream from the specific DNA binding site, resulting in expressionof the reporter gene, such as the lacZ reporter gene. Clones showing thephenotype of the reporter gene expression are selected, and the AD-scFvvectors are isolated. The coding sequences for scFv are identified andcharacterized.

Alternatively, the steps of expressing the library of scFv fusionproteins and expressing the target fusion protein includes causingmating between first and second populations of haploid yeast cells ofopposite mating types. The first population of haploid yeast cellscomprises a library of scFv expression vectors for the library of testerfusion proteins. The second population of haploid yeast cells comprisesa target expression vector. Either the first or second population ofhaploid yeast cells comprises a reporter construct comprising thereporter gene whose expression is under transcriptional control of thetranscription activator.

In this method, the haploid yeast cells of opposite mating types maypreferably be α and a type strains of yeast. The mating between thefirst and second populations of haploid yeast cells of α and a typestrains may be conducted in a rich nutritional culture medium.

FIG. 2B illustrates a flow diagram of a preferred embodiment of theabove described method. As illustrated in FIG. 2B, the sequence librarycontaining scFv fused with an AD domain upstream is carried by a libraryof expression vectors, the AD-scFv vectors. The library of the AD-scFvvectors are transformed into haploid yeast cells such as the a typestrain of yeast.

The coding sequence of the target protein (labeled as “Target”) iscontained in another expression vector and fused with a BD domain,forming the BD-Target vector. The BD-Target vector is transformed intohaploid cells of opposite mating type of the haploid cells containingthe the AD-scFv vectors, such as the α type strain of yeast. Theconstruct carrying the specific DNA binding site and the reporter gene(labeled as “Reporter”) may be transformed into the haploid cells ofeither the type a or type α strain of yeast.

The haploid cells of the type a and type α strains of yeast are matedunder suitable conditions such as low speed of shaking in liquidculture, physical contact in solid medium culture, and rich medium suchas YPD. Bendixen, C. et al. (1994) “A yeast mating-selection scheme fordetection of protein-protein interactions”, Nucleic Acids Res. 22:1778-1779. Finley, Jr., R. L. & Brent, R. (1994) “Interaction matingreveals lineary and ternery connections between Drosophila cell cycleregulators”, Proc. Natl. Acad. Sci. USA, 91:12980-12984. As a result,the AD-scFv, the BD-Target expression vectors and the Reporter constructare taken into the parental diploid cells of the a and type α strain ofhaploid yeast cells.

Upon expression of the sequences in the expression vectors in theparental diploid cells, the library of scFv fusion proteins undergoprotein folding in the host cell and adopt various conformations. Someof the AD-scFv fusion proteins may bind to the Target protein expressedby the BD-Target vector in the parental diploid cell, thereby bringingthe AD and BD domains to a close proximity in the promoter region (i.e.,the specific DNA binding site) of the reporter construct and thusreconstituting a functional transcription activator composed of the ADand BD domains. As a result, the AD activates the transcription of thereporter gene downstream from the specific DNA binding site, resultingin expression of the reporter gene, such as the lacZ reporter gene.Clones showing the phenotype of the reporter gene expression areselected, and the AD-scFv vectors are isolated. The coding sequences forscFv are identified and characterized.

A wide variety of reporter genes may be used in the present invention.Examples of proteins encoded by reporter genes include, but are notlimited to, easily assayed enzymes such as β-galactosidase,α-galactosidase, luciferase, β-glucuronidase, chloramphenicol acetyltransferase (CAT), secreted embryonic alkaline phosphatase (SEAP),fluorescent proteins such as green fluorescent protein (GFP), enhancedblue fluorescent protein (EBFP), enhanced yellow fluorescent protein(EYFP) and enhanced cyan fluorescent protein (ECFP); and proteins forwhich immunoassays are readily available such as hormones and cytokines.The expression of these reporter genes can also be monitored bymeasuring levels of mRNA transcribed from these genes.

When the screening of the scFv library is conducted in yeast cells,certain reporter(s) are of nutritional reporter which allows the yeastto grow on the specific selection medium plate. This is a very powerfulscreening process, as has been shown by many published papers. Examplesof the nutritional reporter include, but are not limited to, His3, Ade2,Leu2, Ura3, Trp1 and Lys2. The His3 reporter is described in Bartel, P.L. et al. (1993) “Using the two-hybrid system to detect protein-proteininteractions”, in Cellular interactions in Development: A practicalapproach, ed. Hastley, D. A., Oxford Press, pages 153-179. The Ade2reporter is described in Jarves, P. et al. (1996) “Genomic libraries anda host strain designed for highly efficient two-hybrid selection inyeast” Genetics 144:1425-1436.

For example, a library of scFv expression vectors that contains a scFvfused with an AD domain of GAL 4 transcription activator (the AD-scFvlibrary) may be transformed into haploid cells of the a mating type ofyeast strain. A BD domain of GAL 4 transcription activator is fused withthe sequence encoding the target protein to be selected against the scFVlibrary in a plasmid. This plasmid is transformed into haploid cells ofthe a mating type of yeast strain.

Equal volume of AD-scFv library-containing yeast stain (α-type) and theBD-target-containing yeast strain (a-type) are inoculated into selectionliquid medium and incubated separately first. These two cultures arethen mixed and allowed to grow in rich medium such as 133 YPD and 2×YPD.Under the rich nutritional culture condition, the two haploid yeaststrains will mate and form diploid cells. At the end of this matingprocess, these yeast cells are plated into selection plates. Amultiple-marker selection scheme may be used to select yeast clones thatshow positive interaction between the scFVs in the library and thetarget. For example, a scheme of SD/-Leu-Trp-His-Ade may be used. Thefirst two selections (Leu-Trp) are for markers (Leu and Trp) expressedfrom the AD-scFv library and the BD-Target vector, respectively. Throughthis dual-marker selection, diploid cells retaining both BD and ADvectors in the same yeast cells are selected. The latter two markers,His-Ade, are used to screen for those clones that express the reportergene from parental strain, presumably due to affinity binding betweenthe scFv in the library and the target.

After the screening by co-transformation, or by mating screening asdescribed above, the putative interaction between the gene probe and thelibrary clone isolates can be further tested and confirmed in vitro orin vivo.

In vitro binding assays may be used to confirm the positive interactionbetween the scFv expressed by the clone isolate and the target peptide.For example, the in vitro binding assay may be a “pull-down” method,such as using GST (glutathione S-transferase)-fused gene probe asmatrix-binding protein, and with in vitro expressed library cloneisolate that are labeled with a radioactive or non-radioactive group.While the probe is bound to the matrix through GST affinity substrate(glutathione-agarose), the library clone isolate will also bind to thematrix through its affinity with the gene probe. The in vitro bindingassay may also be a Co-immuno-precipitation (Co-IP) method using twoaffinity tag antibodies. In this assay, both the target gene probe andthe library clone isolate are in vitro expressed fused with peptidetags, such as HA (haemaglutinin A) or Myc tags. The gene probe is firstimmuno-precipitated with an antibody against the affinity peptide tag(such as HA) that the target gene probe is fused with. Then the secondantibody against a different affinity tag (such as Myc) that is fusedwith the library clone isolate is used for reprobing the precipitate.

In vivo assays may also be used to confirm the positive interactionbetween the scFv expressed by the clone isolate and the target peptide.For example, a mammalian two-hybrid system may serve as a reliableverification system for the yeast two-hybrid library screening. In thissystem, the target gene probe and library clone are fused with Gal 4DNA-binding domain or an mammalian activation domain (such as VP-16)respectively. These two fusion proteins under control of a strong andconstitutive mammalian promoter (such as CMV promoter) are introducedinto mammalian cells by transfection along with a reporter responsive toGal 4. The reporter can be CAT gene (chloramphenical acetatetransferase) or other commonly used reporters. After 2-3 days oftransfection, CAT assay or other standard assays will be performed tomeasure the strength of the reporter which is correlated with thestrength of interaction between the gene probe and the library cloneisolate.

It should be noted that the antibody library described above may bescreened against a target peptide fragment derived from a membraneprotein in other organisms or in vitro. For example, the target peptidemay be expressed as a fusion protein with another protein and screenedagainst the antibody library co-expressed in mammalian cells. The targetpeptide may also be immobilized to a substrate as a single peptide or afusion protein and selected against a library of antibodies displayed onthe surface of bacteriophage or displayed on ribosomes. In addition, thetarget peptide may be introduced to xenomice which contain a library ofhuman antibody and selected for monoclonal human antibodies withspecific binding affinity to target peptide and/or the target membraneprotein.

For example, the library of human antibodies may be screened against atarget peptide derived from a membrane protein (e.g., CCR5) by usingribosome display. Ribosome display is a form of protein display for invitro selection against a target ligand. In this system, mRNA encodingthe tester protein (e.g. an antibody) and the translated tester proteinare associated through the ribosome complex, also called anantibody-ribosome-mRNA (ARM) complex. He and Taussig (1997) Nucleic AcidResearch 25:5132-5134. The principle behind this approach is that singlechain antibody can be functionally produced in an in vitro translationsystem (e.g. rabbit reticulocyte lysate), and in the absence of a stopcodon, individual nascent proteins remain associated with theircorresponding mRNa as stable ternary polypeptide-ribosome-mRNA complexesin such a cell-free system.

In the ribosome display assay, each member of the library of humanantibody sequences includes a bacterial phage T7 promoter and proteinsynthesis initiation sequence attached to the 5′ end of the cDNAencoding the antibody (e.g., scFv) and no stop codon in the 3′ end.Because the cDNA pool is depleted of the stop codon, when the mRNA istranscribed from the cDNA and is subject to in vitro translation, themRNA will still be attached to the ribosome and mRNA, forming the ARMcomplex. The library of human scFv antibody that is translated from thecDNA gene pool and displayed on the surface of the ribosome can bescreened against the target peptide as a single peptide or as a fusionprotein with a protein other than the target membrane protein. The invitro transcription and translation of this library may be carried outin rabbit reticulocyte lysate in the presence of methionine at 30° C. byusing the commercially available systems, such as TNT T7 Quick CoupledTranscription/Translation System (Promega, Madison, Wis.).

The target peptide or its fusion protein may be immobilized to a solidsubstrate, such as a chromatography resin by covalent linkage to enrichfor those ribosomes with high affinity humanized antibody attached. Byaffinity chromatography, the ribosomes with high affinity scFv antibodyattached are isolated. The mRNA encoding the high affinity scFv antibodyis recovered from the isolated ARM complexes and subject to reversetranscriptase (RT)/PCR to synthesize and amplify the cDNA of theselected antibody. This completes the first cycle of the panning processfor antibody isolation and its coding sequence characterization. Such apanning process may be repeated until scFv antibody with desirablyaffinity is isolated.

6. Affinity Maturation of scFv Leads Positively Selected Against TargetPeptide

The binding affinity of the primary scFv antibody leads can be improvedby using an in vitro affinity maturation process according to thepresent invention. The coding sequences of these protein leads may bemutagenized in vitro or in vivo to generated a secondary library morediverse than these leads. The mutagenized leads can be selected againstthe target peptide again in vivo following similar procedures describedfor the selection of the primary library carrying scFv. Such mutagenesisand selection of primary antibody leads effectively mimics the affinitymaturation process naturally occurring in a mammal that producesantibody with progressive increase in the affinity to the immunizingantigen.

The sequences encoding V_(H) and V_(L) of the primary antibody leads aremutagenized in vitro to produce a secondary antibody library. The V_(H)and V_(L) sequences can be randomly mutagenized by “poison” PCR (orerror-prone PCR), by DNA shuffling, or by any other way of random orsite-directed mutagenesis (or cassette mutagenesis). After mutagenesisin the regions of V_(H) and V_(L), the secondary antibody library formedby the mutants of the primary antibody can be screened against thepeptide target by using the yeast two-hybrid system or other screeningmethod. Mutants with higher affinity than the primary antibody lead canbe isolated.

The coding sequences of the scFv leads may be mutagenized by using awide variety of methods. Examples of methods of mutagenesis include, butare not limited to site-directed mutagenesis, error-prone PCRmutagenesis, cassette mutagenesis, random PCR mutagenesis, DNAshuffling, and chain shuffling.

Site-directed mutagenesis or point mutagenesis may be used to graduallychange the V_(H) and V_(L) sequences in specific regions. This isgenerally accomplished by using oligonucleotide-directed mutagenesis.For example, a short sequence of a scFv antibody lead may be replacedwith a synthetically mutagenized oligonucleotide. The method may not beefficient for mutagenizing large numbers of V_(H) and V_(L) sequences,but may be used for fine toning of a particular lead to achieve higheraffinity toward a specific target protein.

Cassette mutagenesis may also be used to mutagenize the V_(H) and V_(L)sequences in specific regions. In a typical cassette mutagenesis, asequence block, or a region, of a single template is replaced by acompletely or partially randomized sequence. However, the maximuminformation content that can be obtained may be statistically limited bythe number of random sequences of the oligonucleotides. Similar to pointmutagenesis, this method may also be used for fine toning of aparticular lead to achieve higher affinity toward a specific targetprotein.

Error-prone PCR, or “poison” PCR, may be used to the V_(H) and V_(L)sequences by following protocols described in Caldwell and Joyce (1992)PCR Methods and Applications 2:28-33. Leung, D. W. et al. (1989)Technique 1:11-15. Shafikhani, S. et al. (1997) Biotechniques23:304-306. Stemmer, W. P. et al. (1994) Proc. Natl. Acad. Sci. USA91:10747-10751.

FIG. 4 illustrates an example of the method of the present invention foraffinity maturation of antibody leads selected from the primary scFvlibrary. As illustrated in FIG. 4, the coding sequences of the scFvleads selected from clones containing the primary scFv library aremutagenized by using a poison PCR method. Since the coding sequences ofthe scFV library are contained in the expression vectors isolated fromthe selected clones, one or more pairs of PCR primers may be used tospecifically amplify the V_(H) and V_(L) region out of the vector. ThePCR fragments containing the V_(H) and V_(L) sequences are mutagenizedby the poison PCR under conditions that favors incorporation ofmutations into the product.

Such conditions for poison PCR may include a) high concentrations ofMn²⁺ (e.g. 0.4-0.6 mM) that efficiently induces malfunction of Taq DNApolymerase; and b) disproportionally high concentration of onenucleotide substrate (e.g., dGTP) in the PCR reaction that causesincorrect incorporation of this high concentration substrate into thetemplate and produce mutations. Additionally, other factors such as, thenumber of PCR cycles, the species of DNA polymerase used, and the lengthof the template, may affect the rate of mis-incorporation of “wrong”nucleotides into the PCR product. Commercially available kits may beutilized for the mutagenesis of the selected scFv library, such as the“Diversity PCR random mutagenesis kit” (catalog No. K1830-1, Clontech,Palo Alto, Calif.).

The PCR primer pairs used in mutagenesis PCR may preferably includeregions matched with the homologous recombination sites in theexpression vectors. This design allows re-introduction of the PCRproducts after mutagenesis back into the yeast host strain again viahomologous recombination. This also allows the modified V_(H) and V_(L)region to be fused with the AD domain directly in the expression vectorin the yeast.

Still referring to FIG. 4, the mutagenized scFv fragments are insertedinto the expression vector containing an AD domain via homologousrecombination in haploid cells of α type yeast strain. Similarly to theselection of scFv clones from the primary antibody library, the AD-scFvcontaining haploid cells are mated with haploid cells of opposite matingtype (e.g. a type) that contains the BD-Target vector and the reportergene construct. The parental diploid cells are selected based onexpression of the reporter gene and other selection criteria asdescribed in detail in Section 5.

Other PCR-based mutagenesis method can also be used, alone or inconjunction with the poison PCR described above. For example, the PCRamplified V_(H) and V_(L) segments may be digested with DNase to createnicks in the double DNA strand. These nicks can be expanded into gaps byother exonucleases such as Bal 31. The gaps may be then be filled byrandom sequences by using DNA Klenow polymerase at low concentration ofregular substrates dGTP, dATP, dTTP, and dCTP with one substrate (e.g.,dGTP) at a disproportionately high concentration. This fill-in reactionshould produce high frequency mutations in the filled gap regions. Thesemethod of DNase I digestion may be used in conjunction with poison PCRto create highest frequency of mutations in the desired V_(H) and V_(L)segments.

The PCR amplified V_(H) and V_(L) segments or the scFv segmentsamplified from the primary antibody leads may be mutagenized in vitro byusing DNA shuffling techniques described by Stemmer (1994) Nature370:389-391; and Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751. The V_(H), V_(L) or scFV segments from the primaryantibody leads are digested with DNase I into random fragments which arethen reassembled to their original size by homologous recombination invitro by using PCR methods. As a result, the diversity of the library ofprimary antibody leads are increased as the numbers of cycles ofmolecular evolution increase in vitro.

The V_(H), V_(L) or scFv segments amplified from the primary antibodyleads may also be mutagenized in vivo by exploiting the inherent abilityof mution in pre-B cells. The Ig gene in pre-B cells is specificallysusceptible to a high-rate of mutation in the development of pre-Bcells. The Ig promoter and enhancer facilitate such high rate mutationsin a pre-B cell environment while the pre-B cells proliferate.Accordingly, V_(H) and V_(L) gene segments may be cloned into amammalian expression vector that contains human Ig enhancer andpromoter. This construct may be introduced into a pre-B cell line, suchas 38B9, which allows the mutation of the V_(H) and V_(L) gene segmentsnaturally in the pre-B cells. Liu, X., and Van Ness, B. (1999) Mol.Immunol. 36:461-469. The mutagenized V_(H) and V_(L) segments can beamplified from the cultured pre-B cell line and re-introduced back intothe AD-containing yeast strain via, for example, homologousrecombination.

The secondary antibody library produced by mutagenesis in vitro (e.g.PCR) or in vivo, i.e., by passing through a mammalian pre-B cell linemay be cloned into an expression vector and screened against the sametarget protein as in the first round of screening using the primaryantibody library. For example, the expression vectors containing thesecondary antibody library may be transformed into haploid cells of αtype yeast strain. These α cells are mated with haploid cells a typeyeast strain containing the BD-target expression vector and the reportergene construct. The positive interaction of scFvs from the secondaryantibody library is screened by following similar procedures asdescribed for the selection of the primary antibody leads in yeast.

Alternatively, since the secondary antibody library may be relativelylow in complexity (e.g., 10⁴-10⁵ independent clones) as compared to theprimary libraries (e.g., 10⁷-10¹⁴), the screening of the secondaryantibody library may be performed without mating between two yeaststrains. Instead, the linearized expression vectors containing the ADdomain and the mutagenized V_(H) and V_(L) segments may be directlyco-transformed into yeast cells containing the BD-target expressionvector and the reporter gene construct. Via homologous recombination inyeast, the secondary antibody library are expressed by the recombinedAD-scFv vector and screened against the target protein expressed by theBD-target vector by following similar procedures as described for theselection of the primary antibody leads in yeast.

7. Functional Expression and Purification of Selected Antibody

The library of scFv fusion protens that are generated and selected inthe screening against the target protein(s) may be expressed in hostsafter the V_(H) and V_(L) sequences are operably linked to an expressioncontrol DNA sequence, including naturally-associated or heterologouspromoters, in an expression vector. By operably linking the V_(H) andV_(L) sequences to an expression control sequence, the V_(H) and V_(L)coding sequences are positioned to ensure the transcription andtranslation of these inserted sequences. The expression vector may bereplicable in the host organism as episomes or as an integral part ofthe host chromosomal DNA. The expression vector may also containselection markers such as antibiotic resistance genes (e.g. neomycin andtetracycline resistance genes) to permit detection of those cellstransformed with the expression vector.

Preferably, the expression vector may be a eukaryotic vector capable oftransforming or transfecting eukaryotic host cells. Once the expressionvector has been incorporated into the appropriate host cells, the hostcells are maintained under conditions suitable for high level expressionof the single-chains polypeptide encoded by a scFv. The polypeptidesexpressed are collected and purified depending on the expression systemused.

The scFv, Fab, or fully assembled antibodies selected by using themethods of the present invention may be expressed in various scales inany host system such as bacteria (e.g. E. coli), yeast (e.g. S.cerevisiae), and mammalian cells (COS). The bacteria expression vectormay preferably contain the bacterial phage T7 promoter and express asingle chain variable fragment (scFv). The yeast expression vector maycontain a constitutive promoter (e.g. ADGI promoter) or an induciblepromoter such as (e.g. GCN4 and Gal 1 promoters). All three types ofantibody, scFv, Fab, and full antibody, may be expressed in a yeastexpression system.

The expression vector may be a mammalian express vector that can be usedto express the single-chains polypeptide encoded by V_(H) and V_(L) inmammalian cell culture transiently or stably. Examples of mammalian celllines that may be suitable of secreting immunoglobulins include, but arenot limited to, various COS cell lines, HeLa cells, myeloma cell lines,CHO cell lines, transformed B-cells and hybridomas.

Typically, a mammalian expression vector includes certain expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, as well as necessary processing signals, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Examples of promoters include, butare not limited to, insulin promoter, human cytomegalovirus (CMV)promoter and its early promoter, simian virus SV40 promoter, Roussarcoma virus LTR promoter/enhancer, the chicken cytoplasmic β-actinpromoter, promoters derived from immunoglobulin genes, bovine papillomavirus and adenovirus.

One or more enhancer sequence may be included in the expression vectorto increase the transcription efficiency. Enhancers are cis-actingsequences of between 10 to 300 bp that increase transcription by apromoter. Enhancers can effectively increase transcription whenpositioned either 5′ or 3′ to the transcription unit. They may also beeffective if located within an intron or within the coding sequenceitself. Examples of enhancers include, but are not limited to, SV40enhancers, cytomegalovirus enhancers, polyoma enhancers, the mouseimmunoglobulin heavy chain enhancer. and adenovirus enhancers. Themammalian expression vector may also typically include a selectablemarker gene. Examples of suitable markers include, but are not limitedto, the dihydrofolate reductase gene (DHFR), the thymidine kinase gene(TK), or prokaryotic genes conferring antibiotic resistance. The DHFRand TK genes prefer the use of mutant cell lines that lack the abilityto grow without the addition of thymidine to the growth medium.Transformed cells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The expression vectors containing the scFv sequences can then betransferred into the host cell by methods known in the art, depending onthe type of host cells. Examples of transfection techniques include, butare not limited to, calcium phosphate transfection, calcium chloridetransfection, lipofection, electroporation, and microinjection.

The V_(H) and V_(L) sequences may also be inserted into a viral vectorsuch as adenoviral vector that can replicate in its host cell andproduce the polypeptide encoded by V_(H) and V_(L) in large amounts.

In particular, the scFv, Fab, or fully assembled antibody may beexpressed in mammalian cells by using a method described by Persic etal. (1997) Gene, 187:9-18. The mammalian expression vector that isdescribed by Persic and contains EF-α promoter and SV40 replicationorigin is preferably utilized. The SV40 origin allows a high level oftransient expression in cells containing large T antigen such as COScell line. The expression vector may also include secretion signal anddifferent antibiotic markers (e.g. neo and hygro) for integrationselection.

Once expressed, polypeptides encoded by V_(H) and V_(L) may be isolatedand purified by using standard procedures of the art, including ammoniumsulfate precipitation, fraction column chromatography, and gelelectrophoresis. Once purified, partially or to homogeneity as desired,the polypeptides may then be used therapeutically or in developing,performing assay procedures, immunofluorescent stainings, and in otherbiomedical and industrial applications. In particular, the antibodiesgenerated by the method of the present invention may be used fordiagnosis and therapy for the treatment of various diseases such ascancer, autoimmune diseases, or viral infections.

In a preferred embodiment, the scFv human antibody with V_(H) and V_(L)segments that are generated and screened by using the methods of thepresent invention may be expressed directly in yeast. According to thisembodiment, the V_(H) and V_(L) regions from the selected expressionvectors may be PCR amplified with primers that simultaneously addappropriate homologous recombination sequences to the PCR products.These PCR segments of V_(H) and V_(L) may then be introduced into ayeast strain together with a linearized expression vector containingdesirable promoters, expression tags and other transcriptional ortranslational signals.

For example, the PCR segments of V_(H) and V_(L) regions may behomologously recombined with a yeast expression vector that alreadycontains a desirable promoter in the upstream and stop codons andtranscription termination signal in the downstream. The promoter may bea constitutive expression promoter such as ADH1, or an inducibleexpression promoter, such as Gal 1, or GCN4 (A. Mimran, I. Marbach, andD. Engelberg, (2000) Biotechniques 28:552-560). The latter induciblepromoter may be preferred because the induction can be easily achievedby adding 3-AT into the medium.

The yeast expression vector to be used for expression of the scFvantibody may be of any standard strain with nutritional selectionmarkers, such as His 3, Ade 2, Leu 2, Ura 3, Trp 1 and Lys 2. The markerused for the expression of the selected scFv may preferably be differentfrom the AD vector used in the selection of scFv in the two-hybridsystem. This may help to avoid potential carryover problem associatedwith multiple yeast expression vectors.

For expressing the scFv antibody in a secreted form in yeast, theexpression vector may include a secretion signal in the 5′ end of theV_(H) and V_(L) segments, such as an alpha factor signal and a 5-phosecretion signal. Certain commercially available vectors that contain adesirable secretion signal may also be used (e.g., pYEX-S1, catalog#6200-1, Clontech, Palo Alto, Calif.).

The scFv antibody fragments generated may be analyzed and characterizedfor their affinity and specificity by using methods known in the art,such as ELISA, western, and immune staining. Those scFv antibodyfragments with reasonably good affinity (with dissociation constantpreferably above 10⁻⁶ M) and specificity can be used as building blocksin Fab expression vectors, or can be further assembled with the constantregion for full length antibody expression. These fully assembled humanantibodies may also be expressed in yeast in a secreted form.

The V_(H) sequence encoding the selected scFv protein may be linked withthe constant regions of a full antibody, C_(H)1, C_(H)2 and C_(H)3.Similarly, the V_(L) sequence may be linked with the constant regionC_(L). The assembly of two units of V_(H)-C_(H)1-C_(H)2-C_(H)3 andV_(L)-C_(L)-C_(L) leads to formation of a fully functional antibody. Thepresent invention provides a method for producing fully functionalantibody in yeast. Fully functional antibody retaining the rest of theconstant regions may have a higher affinity (or avidity) than a scFv ora Fab. The full antibody should also have a higher stability, thusallowing more efficient purification of antibody protein in large scale.

The method is provided by exploiting the ability of yeast cells touptake and maintain multiple copies of plasmids of the same replicationorigin. According to the method, different vectors may be used toexpress the heavy chain and light chain separately, and yet allows forthe assembly of a fully functional antibody in yeast. This approach hasbeen successfully used in a two-hybrid system design where the BD and ADvectors are identical in backbone structure except the selection markersare distinct. This approach has been used in a two-hybrid system designfor expressing both BD and AD fusion proteins in the yeast. The BD andAD vectors are identical in their backbone structures except theselection markers are distinct. Both vectors can be maintained in yeastin high copy numbers. Chien, C. T., et al. (1991) “The two-hybridsystem: a method to identify and clone genes for proteins that interactwith a protein of interest” Proc. Natl. Acad. Sci. USA 88:9578-9582.

In the present invention, the heavy chain gene and light chain genes areplaced in two different vectors. Under a suitable condition, theV_(H)-C_(H)1-C_(H)2-C_(H)3 and V_(L)-C_(L) sequences are expressed andassembled in yeast, resulting in a fully functional antibody proteinwith two heavy chains and two light chains. This fully functionalantibody may be secreted into the medium and purified directly from thesupernatant.

The scFv with a constant region, Fab, or fully assembled antibody can bepurified using methods known in the art. Conventional techniquesinclude, but are not limited to, precipitation with ammnonium sulfateand/or caprylic acid, ion exchange chromatography (e.g. DEAE), and gelfiltration chromatography. Delves (1997) “Antibody Production: EssentialTechniques”, New York, John Wiley & Sons, pages 90-113. Affinity-basedapproaches using affinity matrix based on Protein A, Protein G orProtein L may be more efficiency and results in antibody with highpurity. Protein A and protein G are bacterial cell wall proteins thatbind specifically and tightly to a domain of the Fc portion of certainimmunoglobulins with differential binding affinity to differentsubclasses of IgG. For example, Protein G has higher affinities formouse IgG1 and human IgG3 than does Protein A. The affinity of Protein Aof IgG1 can be enhanced by a number of different methods, including theuse of binding buffers with increased pH or salt concentration. ProteinL binds antibodies predominantly through kappa light chain interactionswithout interfering with the antigen-binding site. Chateau et al. (1993)“On the interaction between Protein L and immunoglobulins of variousmammalian species” Scandinavian J. Immunol., 37:399-405. Protein L hasbeen shown to bind strongly to human kappa light chain subclasses I, IIIand IV and to mouse kappa chain subclasses I. Protein L can be used topurify relevant kappa chain-bearing antibodies of all classes (IgG, IgM,IgA, IgD, and IgE) from a wide variety of species, including human,mouse, rat, and rabbit. Protein L can also be used for the affinitypurification of scFv and Fab antibody fragments containing suitablekappa light chains. Protein L-based reagents is commercially availablefrom Actigen, Inc., Cambridgem, England. Actigen can provide a line ofrecombinant Protein products, including agarose conjugates for affinitypurification and immobilized forms of recombinant Protein L and A fusionprotein which contains four protein A antibody-binding domains and fourprotein L kappa-binding domains.

Other affinity matrix may also be used, including those that exploitpeptidomimetic ligands, anti-immunoglobulins, mannan binding protein,and the relevant antigen. Peptidomimetic ligands resemble peptides butthey do not correspond to natural peptides. Many of Peptidomimeticligands contain unnatural or chemically modified amino acids. Forexample, peptidomimetic ligands designed for the affinity purificationof antibodies of the IGA and IgE classes are commercially available fromTecnogen, Piana di Monte Verna, Italy. Mannan binding protein (MBP) is amannose- and N-acetylglucosamine-specific lectin found in mammaliansera. This lectin binds IgM. The MBP-agarose support for thepurification IgM is commercially available from Pierce.

Immunomagnetic methods that combine an affinity reagent (e.g. protein Aor an anti-immunoglobulin) with the ease of separation conferred byparamagnetic beads may be used for purifying the antibody produced.Magnetic beads coated with Protein or relevant secondary antibody may becommercially available from Dynal, Inc., NY; Bangs Laboratories,Fishers, Ind.; and Cortex Biochem Inc., San Leandro, Calif.

Direct expression and purification of the selected antibody in yeast isadvantageous in various aspects. As a eukaryotic organism, yeast is moreof an ideal system for expressing human proteins than bacteria or otherlower organisms. It is more likely that yeast will make the scFv, Fab,or fully assembled antibody in a correct conformation (foldedcorrectly), and will add post-translation modifications such as correctdisulfide bond(s) and glycosylations.

Yeast has been explored for expressing many human proteins in the past.Many human proteins have been successfully produced from the yeast, suchas human serum albumin (Kang, H. A. et al. (2000) Appl. Microbiol.Biotechnol. 53:578-582) and human telomerase protein and RNA complex(Bachand, F., et al. (2000) RNA 6:778-784).

Yeast has fully characterized secretion pathways. The genetics andbiochemistry of many if not all genes that regulate the pathways havebeen identified. Knowledge of these pathways should aid in the design ofexpression vectors and procedures for isolation and purification ofantibody expressed in the yeast.

Moreover, yeast has very few secreted proteases. This should keep thesecreted recombinant protein quite stable. In addition, since yeast doesnot secrete many other and/or toxic proteins, the supernatant should berelatively uncontaminated. Therefore, purification of recombinantprotein from yeast supernatant should be simple, efficient andeconomical.

Additionally, simple and reliable methods have been developed forisolating proteins from yeast cells. Cid, V. J. et al. (1998) “Amutation in the Rho&GAP-encoding gene BEM2 of Saccharomyces cerevisiaeaffects morphogenesis and cell wall functionality” Microbiol. 144:25-36.Although yeast has a relatively thick cell wall that is not present ineither bacterial or mammalian cells, the yeast cells can still keep theyeast strain growing with the yeast cell wall striped from the cells. Bygrowing the yeast strain in yeast cells without the cell wall, secretionand purification of recombinant human antibody may be made more feasibleand efficient.

By using yeast as host system for expression, a streamlined process canbe established to produce recombinant antibodies in fully assembled andpurified form. This may save tremendous time and efforts as compared tousing any other systems such as humanization of antibody in vitro andproduction of fully human antibody in transgenic animals.

In summary, the compositions, kits and methods provided by the presentinvention should be very useful for selecting proteins such as humanantibodies with high affinity and specificity against a wide variety oftargets including, but not limited to, soluble proteins (e.g. growthfactors, cytokines and chemokines), membrane-bound proteins (e.g. cellsurface receptors), and viral antigens. The whole process of libraryconstruction, functional screening and expression of highly diverserepertoire of human antibodies can be streamlined, and efficiently andeconomically performed in yeast in a high throughput and automatedmanner. The selected proteins can have a wide variety of applications.For example, they can be used in therapeutics and diagnosis of diseasesincluding, but not limited to, autoimmune diseases, cancer, transplantrejection, infectious diseases and inflammation.

EXAMPLE

1. Construction of Human Single Chain Antibody Library

A human scFv library was constructed in a yeast two-hybrid vector pACT2that contains sequence encoding Gal4 activation domain (AD) (Li et al.(1994) “Specific association between the human DNA repair proteins XPAand ERCC1” Proc Natl Acad Sci U S A. 91:5012-5016). cDNA encoding thevariable regions of heavy (V_(H)) and light chain (V_(L)) were amplifiedby RT-PCR from poly A⁺ RNA of human spleen, bone marrow, fetal liver andperipheral blood leukocytes (PBL). The V_(H) and V_(L) cDNA fragmentswere linked by a linker encoding [(Gly)₄Ser]₄ (Nicholls et al. (1993)“An improved method for generating single-chain antibodies fromhybridomas” J Immunol Methods 165:81-91), and are flanked by sequencesof approximately 60 bp at each end that are homologous to the pACT2multiple cloning sites (MCS) (Hua, et al, (1998) “Construction of amodular yeast two-hybrid cDNA library from human EST clones for thehuman genome protein linkage map” Gene. 215:143-152). Such assembled PCRproducts were cloned into pACT2 by homologous recombination (Hua et al,1997) in yeast cells (MATα strains Y187 or MaV203) (Harper et al, (1993)“The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1cyclin-dependent kinases” Cell 75:805-16; Vidal et al. (1996) “Reversetwo-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions” Proc Natl Acad Sci U S A.93:10315-10320). Such derived human scFvs are fused in-frame with theGal4 activation domain. A total of 5×10⁷ independent yeast colonies wereharvested and stored at −80° C.

More specifically, poly A⁺ RNA from human bone marrow, human fetalliver, human spleen and human peripheral blood leukocytes were purchasedfrom Clontech Laboratories (Palo Alto, Calif.). First strand cDNA weremade from the poly A⁺ RNA using random primer and PowerScript reversetranscriptase kit (Clontech Laboratories, Palo Alto, Calif.). A set ofoligonucleotides designed by Sblattero and Bradbury (Sblattero andBradbury (1998) “A definitive set of oligonucleotide primers foramplifying human V regions” Immunotechnology. 3:271-278) that recognizeall functional V genes were used to amplify all variable regions ofheavy chain and light chain of human antibodies in PCR (Marks et al.(1991) “By-passing immunization: Human antibodies from V-gene librariesdisplayed on phage” J Mol Biol. 222:581-597).

The cDNA of heavy chain variable region (V_(H)) and light chain variableregion (V_(L)) were linked by a short linker sequence encoding[(Gly)₄Ser]₄ (5′-GGC GGT GGT GGA TCA GGC GGC GGA GGA TCT GGC GGA GGT GGCAGC GGT GGT GGA GGC AGT-3′ [SEQ ID NO: 5]) (Nicholls et al. (1993) “Animproved method for generating single-chain antibodies from hybridomas”J Immunol Methods 165:81-91). The V_(H)-linker-V_(L) cassettes wereflanked by 60 base pairs (bp) at its 5′ end and 57 bp at its 3′ end ofsequence homologous to the sequence adjacent to multiple cloning site ofthe yeast two-hybrid vector pACT2 (Hua et al, (1997), supra, Hua et al(1998), supra).

The 5′ (1.3.a) and 3′ homologous sequence (1.3.b) are as follows:

[SEQ ID NO: 6] 1.3.a: 5′-ACC CCA CCA AAC CCA AAA AAA GAG ATC TGT ATG GCTTAC CCA TAC GAT GTT CCA GAT TAC [SEQ ID NO: 7] 1.3.b: 5′-GAG ATG GTG CACGAT GCA CAG TTG AAG TGA ACT TGC GGG GTT TTT CAG TAT CTA CGA

The above-assembled PCR products containing scFv were co-transformedwith linearized pACT2 DNA (Hua et al. (1997), supra) into yeast strainsY187 (MATα, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3,112,gal4 Δ, gal80Δ, URA3::GAL1_(UAS)-GAL1_(TATA)-lacZ) (Harper et al, 1993)or MaV203 (MATα, ura3-52, his3Δ200, ade2-101, trp1-901, leu2-3,112,cyh2^(R), can1^(R), gal4Δ, gal80Δ, GAL1::lacZ,HIS3_(UASGAL1)::HIS3@LYS2, SPAL10::URA3) (Vidal et al. (1996) “Reversetwo-hybrid and one-hybrid systems to detect dissociation ofprotein-protein and DNA-protein interactions” Proc Natl Acad Sci U S A.3:10315-10320). The transformants were plated on yeast synthetic mediumlacking leucine (SD/-L) and incubated at 30° C. for 2 days. A total ofapproximately 5×10⁷ independent colonies of the yeast two-hybrid scFvlibrary were harvested and stored at −80° C.

2. Construction of a Yeast Expression Vector Encoding Peptide FragmentsDerived from Human CCR5

Peptide fragments derived from human CCR5 were used as target peptidesagainst which the scFv library constructed above was screened. Threeextracellular domains of human CCR5 cDNA, an N-terminal fragment, the4^(th) loop (or loop 4) and the 6^(th) loop (or loop 6), were separatelyPCR-amplified from human leukocyte cDNA (Clontech Laboratories, Inc.,Palo Alto, Calif.) using the following oligonucleotide primers.

For amplification of the N-terminus of human CCR5 (aa 1-36), the primerpair are:

[SEQ ID NO: 49] 13.13.L 5′-GGA GAA TTC GATTATCAAGTGTCAAGTCCA [SEQ ID NO:50] 13.13.M 5′-CGC GGA TCC TTA GAGCGGAGGCAGGAGGCGG

Primer 13.13.L corresponds to the N-terminus of CCR5, with an Eco R1site added. Primer 13.13.M complements the sequence at the end ofN-terminal extracellular domain (aa 36) of CCR5, with Bam HI and Stopcodon added.

For amplification of the 4^(th) loop of human CCR5 (aa 167-198), theprimer pair are:

[SEQ ID NO: 51] 13.13.N 5′-GGA GAA TTC ACCAGATCTCAAAAAGAAGG [SEQ ID NO:52] 13.13.O 5′-CGC GGA TCC TTA TATCTTTAATGTCTGGAAATT

Primer 13.13.N corresponds the sequence at the N-terminus of 4^(th) loopof CCR5 (aa 167), with Eco RI site added. Primer 13.13.0 complements thesequence at the C-terminus of 4^(th) loop of CCR5 (aa 198), with Bam HIand Stop codon added.

For amplification of the 6^(th) loop of CCR5 (aa 262-290), the primerpair are:

[SEQ ID NO: 53] 13.13.P 5′-CAG GAA TTC TTTGGCCTGAAT [SEQ ID NO: 54]13.13.Q 5′-CGC GGA TCC TCA GCAGTGCGTCATCCCAAGA

Primer 13.13.P corresponds the sequence at the N-terminus of 6^(th) loopof hCCR5 (aa 262) at the Eco RI site. Primer 13.13.Q complements thesequence at the C-terminus of 6^(th) loop of CCR5 (aa 290), with Bam HIand Stop codon added.

The PCR product of each of the domains was cloned into an Eco RI/BamHI-digested cloning vector pGBKT7 (Clontech Laboratories, Palo Alto,Calif.) with the Gal4 DNA binding domain (DNA-BD) at its carboxyterminus. The resulting plasmid were designated as follows:

pG90: pGBKT7-CCR5 N-terminus;

pG91: pGBKT7-CCR5 loop 4;

pG92: pGBKT7-CCR5 loop 6.

Each of the above plasmids encoding CCR5 peptide fragments wastransformed into yeast strain AH109 (MATa, ura3-52, his3-200, ade2-101,trp1-901, leu2-3,112, gal4Δ, gal80Δ, LYS2::GAL1_(UAS)-GAL1_(TATA)-HIS3,GAL2_(UAS)-GAL2_(TATA)-ADE2, URA3::MEL1_(UAS)-MEL1_(TATA)-lacZ)(Clontech Laboratories, Palo Alto, Calif.). The transformants wereselected on synthetic medium lacking tryptophan (SD/-W).

3. Screening of a Human scFv Library Against Extracellular Domains ofHuman CCR5

To screen the scFv library against the extracellular domains of humanCCR5, the AH 109 transformants containing one of the three extracellulardomains were mated with MATα type yeast cells (Y187 or MaV203 strain)containing the scFv library following the protocols from ClontechLaboratories. The scFv library-containing vector pACT2 contains a LEU2gene, whereas the pGBKT7 plasmids contain a TRP1 gene. Cells harboringboth plasmids can grow in the yeast synthetic medium lacking leucine andtryptophan (SD/-LW). Interactions between a scFv and the target CCR5domain activated expression of reporter genes ADE2 and HIS3 built ingenome of the strains, thus allowing the cells to grow on medium lackingadenine, histidine, leucine and tryptophan (SD/-AHLW). Colonies thatwere able to grow on SD/-ALHW medium were picked. These colonies wereassayed for the expression of additional reporter gene lacZ in theβ-galactosidase colony-lifting assay as described in the instructionmanual from Clontech Laboratories. Plasmid DNA of pACT2 containing thescFv fragment was retrieved from the yeast cells.

To analyze the specificity of those scFv clones isolated from theabove-mentioned library screening, pGBKT7 plasmids encoding CCR5domains, empty vector pGBKT7 and pGBKT7-Lam (Clontech) (which containssequence of human lamin C), were co-transformed, respectively, withindividual scFv plasmids into yeast cells, followed by growth selectionon SD/-LW or SD/-AHLW media. Yeast colonies grown on the selection mediawere subjected to β-galactosidase activity assays. The sequences ofthose scFv clones specific to human CCR5 domains were determined with anABI automatic sequencer.

From the above library screening and specificity analysis, one specificscFv clone (clone 15.186.35) was obtained against the N-terminalfragment of human CCR5, and 3 specific scFv clones against Loop 6 ofhuman CCR5: clones 15.150.11, 15.150.12, and 15.150.24.

The DNA and amino acid sequences encoding these four clones are listedin FIG. 5. In addition, some variants of the four clones with slightmodifications in the sequences in the framework regions are listed inFIG. 6.

4. Inhibition of HIV-1 Infection by the Selected Human MonoclonalAntibody

ScFv clones 15.150.11 and 15.150.12 were cloned into E. coli expressionvector pET27b(+) (Novagen) to facilitate expression of scFv antibodiesof Ab32 and Ab33, respectively. ScFv proteins were expressed andpurified from the periplasmic space of the bacteria. The ability of theselected anti-CCR5 scFv antibodies, Ab32 and Ab33, to inhibit HIV-1infection was determined by using an assay described in Cotter et al.(2001) J. Virol. 75:4308-4320. The control scFv antibody used wasanti-human p53 scFv antibody.

Human monocytes were recovered from peripheral blood mononuclear cellsof HIV-1-, HIV-2-, and hepatitis B virus-seronegative donors afterleukapheresis and then purified by countercurrent centrifugalelutriation. Monocytes were cultured as adherent monolayers anddifferentiated for 7 days into macrophages (monocyte-derived macrophagesor MDM). MDM were first incubated with different concentrations of scFvantibodies, then infected with HIV-1. HIV-1 reverse transcriptase (RT)activity was determined at Day 4, Day 8 and Day 12, as incorporation of[³H]TTP (Cotter et al, 2001, J. Virol. 75:4308-4320). Radiolabelednucleotides were precipitated with cold 10% trichloroacetic acid onpaper filters in an automatic cell harvester and washed with 95%ethanol. Radioactivity was estimated by liquid scintillationspectroscopy. The cell viability was determined by MTT assay. Briefly,MTT (3-{4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, fromSigma) was dissolved in cell culture medium without phenol red. Livecells will convert MTT into purple dye inside cells. The dye wassolubilized with acidic isopropanol and absorbance (OD) of the converteddye was measured at 570 nm. Mossman T., 1983, J. Immunol. Methods 65:55.

FIGS. 9A-C show HIV-1 RT activity in monocytes infected by HIV-1 in thepresence or absence of two selected scFv antibodies against human CCR5Loop (Ab32 and AB33) of the present invention on day 4, 8, and 12 postinfection, respectively. As shown in FIGS. 9A-C, both Ab32 and AB33effectively inhibit HIV-1 RT activity at concentrations 20, 2.0 and 0.2μg/mL. In contrast, a non-specific antibody which is elicited againstthe tumor suppressor p53 protein), Ab 16, is completely ineffective ininhibition of HIV-1 RT activity. The positive control, a murinemonoclonal antibody 2D7 (available from Pharmingen, San Diego, Calif.)could inhibit HIV-1 RT activity at a concentration of 10 μg/mL. In theabsence of antibodies or with addition of mere buffer the HIV RTactivity is completely uninhibited. Throughout the incubation period,the HIV-infected monocytes had normal viability in the presence orabsence of these antibodies, as shown in FIGS. 10A-C.

More significantly, when the concentrations of both Ab32 and AB33 werelowered to 0.02 μg/mL, these two scFv antibodies were still effectivelyinhibit inhibit HIV-1 RT activity. FIGS. 11A-C show HIV-1 RT activity inmonocytes infected by HIV-1 in the presence or absence of Ab32 and AB33at various concentrations on day 4, 8, and 12 post infection,respectively. At a concentration as lower as 0.02 μg/mL (˜0.8 nM), bothAb32 and AB33 could inhibit HIV-1 RT activity by 75% on day 12 postinfection of the monocytes.

5. Binding of the Selected Human Monoclonal Antibody to CCR5

The ability of the human monoclonal scFv antibodies Ab32 and Ab33 tobind with their target protein was confirmed by Western blot. Briefly,lysate of human macrophage (expressing CCR5) was separated on SDS-PAGE,and transferred to nitrocellulose membrane. The membrane was then probedeither with the scFv selected in the above-described process (Ab32 andAb33) or positive control antibody (murine monoclonal antibody 2D7 fromPharmingen, San Diego), or a negative control (Ab16, an anti-p53 scFvantibody). The positive control (MAb 2D7) blot was then probed with goatanti-mouse IgG conjugated with HRP (horse radish peroxidase). ThescFv-probed blots were incubated with mouse anti-HSV tag antibodyfollowed by goat anti-mouse IgG conjugated HRP. The CCR5 band was thendetected with ECL (Enhanced Chemilluminence, from Amersham-Pharmacia).

FIG. 12 shows the Western blot of CCR5 expressed by human macrophageprobed by Ab32 and Ab33. As shown in FIG. 12, both Ab32 and Ab33 werecapable of binding to CCR5, just like the positive control MAb 2D7. Incontrast, a non-specific scFv antibody elicited against human p53protein, AB16, is incapable of binding to CCR5.

These results indicate that the monoclonal scFv antibodies selectedagainst a peptide fragment derived from CCR5 Loop 6 can specificallyrecognize and bind to human CCR5 in vitro.

6. Inhibition of Chemokines Binding to to CCR5 by the Selected HumanMonoclonal Antibody

The ability of the human monoclonal scFv antibodies Ab32 and Ab33 tobind with their target protein was further validated by conductingcompetition binding assay using described in Wu et al. (1997) J. Exp.Med. 186:1373-1381. Briefly, human MDMs (monocyte-derived macrophages)were plated in 48-well plates. The attached cells were incubated withantibodies (Ab32, Ab33, or the mouse monoclonal antibody 2D7) at 37° C.for 30 minutes. Radio-labeled human CCR5 ligand, ¹²⁵I MIP1-α (Amersham),was added to each well to a final concentration of 100 pM (2 μCi/pmole)and the cultures were incubated at 37° C. for two hours. After removalof the medium, the cultures were washed 3 times with cold PBS buffer.Cells were lysed with 0.3 ml of 1% Triton X-100 in PBS for 30 min atroom temperature. Radioactivity in the lysates were measured by a gammacounter (Packard). The results were shown in FIG. 13.

As shown in FIG. 13, the human monoclonal scFv antibodies Ab32 and Ab33effectively blocked the binding of ₁₂₅I MIP1-α to its cognate receptorCCR5 on human MDMs. Significantly, both Ab32 and Ab33 exhibited slightlystronger binding affinity to human CCR5 than the mouse monoclonalantibody 2D7. In contrast, a non-specific human scFv against human p53,Ab16, could not inhibit the binding of ¹²⁵I MIP1-α to CCR5.

To ensure that the results obtained in above-described assay wereobtained in a normally-behaving binding assay, non-labeled MIP1-α wasused to compete with ¹²⁵I MIP1-α for binding to CCR5. As shown in FIG.14, MIP1-α could compete with ¹²⁵I MIP1-α for binding to CCR5 at aconcentration of 25 nm. Similarly, another cognate ligand of human CCR5,RANTES, could also compete with ¹²⁵I MIP1-α for binding to CCR5 at aconcentration of 25 nm. These results indicate that the radio-labeledhuman CCR5 ligand, ¹²⁵I MIP1-α, did bind to its cognate receptor CCR5 onhuman MDMs and the binding could be inhibited by the human monoclonalscFv antibodies Ab32 and Ab33 selected using the method of the presentinvention.

1. An isolated antibody that specifically binds to human CCR5, whereinthe antibody comprises a heavy chain variable region of SEQ ID NO: 55and a light chain variable region of SEQ ID NO: 58, a heavy chainvariable region of SEQ ID NO: 56 and a light chain variable region ofSEQ ID NO: 59, or a heavy chain variable region of SEQ ID NO: 57 and alight chain variable region of SEQ ID NO:
 60. 2. The antibody of claim1, wherein the antibody specifically binds to loop 6 of human CCR5 andinhibits infection of immunodeficiency virus of human cells.
 3. Theantibody of claim 1, wherein the antibody is a monoclonal antibody. 4.The antibody of claim 3, wherein the monoclonal antibody is a singlechain antibody.
 5. The antibody of claim 4, wherein the single chainantibody comprises an amino acid sequence selected from SEQ ID Nos: 27,29, and
 31. 6. The antibody of claim 4, wherein the antibody has anamino acid sequence of SEQ ID NO:
 27. 7. The antibody of claim 4,wherein the antibody is encoded by a polynucleotide selected from thegroup consisting of SEQ ID NOs: 26, 28, and
 30. 8. The antibody of claim1, wherein the antibody specifically binds to loop 6 of human CCR5 whichcomprises SEQ ID NO:
 2. 9. A recombinant expression vector encoding apolypeptide selected from the group consisting of SEQ ID NOs: 36-41. 10.The recombinant expression vector of claim 9 is a bacterial, yeast,plant, mammalian or viral expression vector.
 11. A recombinant cellexpressing a polypeptide selected from the group consisting of SEQ IDNOs: 36-41.
 12. The recombinant cell of claim 11 is a bacterial, yeast,plant or mammalian cell.
 13. The recombinant cell of claim 11 is a humancell.